NZ615328B2 - Fusion proteins and combination vaccines comprising haemophilus influenzae protein e and pilin a - Google Patents
Fusion proteins and combination vaccines comprising haemophilus influenzae protein e and pilin a Download PDFInfo
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- NZ615328B2 NZ615328B2 NZ615328A NZ61532812A NZ615328B2 NZ 615328 B2 NZ615328 B2 NZ 615328B2 NZ 615328 A NZ615328 A NZ 615328A NZ 61532812 A NZ61532812 A NZ 61532812A NZ 615328 B2 NZ615328 B2 NZ 615328B2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/04—Antibacterial agents
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- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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Abstract
Disclosed is a fusion protein of formula I: (X)m - (R1)n - A - (Y)o - B - (Z)p (formula I) wherein: X is a signal peptide or MHHHHHH; m is 0 or 1; R1 is an amino acid; n is 0, 1, 2, 3, 4, 5 or 6; A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, or PilA from Haemophilus influenzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h wherein h is 4, 5, 6, 7, 8,9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment thereof, or Protein E from Haemophilus influenzae or an immunogenic fragment thereof; Z is GGHHHHHH (SEQ 10 NO.3); and p is 0 or 1, wherein when A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an immunogenic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an immunogenic fragment thereof, B is not PilA from Haemophilus influenzae or an immunogenic fragment thereof. Also disclosed is an immunogenic composition comprising isolated Protein E from H. influenzae and isolated PilA from H. influenzae ophilus influenzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h wherein h is 4, 5, 6, 7, 8,9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment thereof, or Protein E from Haemophilus influenzae or an immunogenic fragment thereof; Z is GGHHHHHH (SEQ 10 NO.3); and p is 0 or 1, wherein when A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an immunogenic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an immunogenic fragment thereof, B is not PilA from Haemophilus influenzae or an immunogenic fragment thereof. Also disclosed is an immunogenic composition comprising isolated Protein E from H. influenzae and isolated PilA from H. influenzae
Description
/050236
FUSION PROTEINS AND COMBINATION VACCINES COMPRISING HAEMOPHILUS
INFLUENZAE PROTEIN E AND PILIN A
This application claims priority to United States patent application number 779
filed April 13, 2011 and United States patent application number 61/534012 filed September 13,
2011.
FIELD OF THE ION
The present invention relates to itions sing Haemophi/us influenzae (H.
influenzae) Protein E and Pilin A. More particularly, the present application relates to fusion
proteins and immunogenic compositions comprising Protein E and Pilin A, vaccines
comprising such immunogenic compositions and therapeutic uses of the same.
BACKGROUND OF THE INVENTION
Protein E (PE) is an outer membrane lipoprotein with adhesive ties. It plays a
role in the adhesion/invasion of peable Haemophi/us influenzae (NTHi) to epithelial
cells. (J. Immunology 183: 2593-2601 (2009); The Journal of Infectious Diseases 2-531
(2009), Microbes and Infection 10:87-96 (2008)). It is highly conserved in both encapsulated
Haemophi/us influenzae and non-typeable H. influenzae and has a conserved epithelial
binding . (The Journal of ious Diseases 201:414-419 (2010)). Thirteen different
point mutations have been described in different Haemophi/us species when compared with
Haemophi/us influenzae Rd as a reference strain. Its expression is observed on both
logarithmic growing and stationary phase bacteria. (WO2007/084053).
Protein E is also involved in human complement ance through binding vitronectin.
(Immunology 183: 2593-2601 (2009)). PE, by the binding domain PKRYARSVRQ
YKILNCANYH LTQVR (SEQ ID NO. 1, corresponding to amino acids 84-108 of SEQ ID NO.
4), binds vitronectin which is an important inhibitor of the terminal complement pathway. (J.
Immunology 93-2601 (2009)).
Pilin A (PiIA) is likely the major pilin subunit of H. influenzae Type IV Pilus (Tfp) involved
in ing motility (Infection and Immunity, 73: 1635-1643 (2005)). NTHi PilA is a conserved
adhesin expressed in vivo. It has been shown to be involved in NTHi adherence, colonization
and biofilm formation. ular Microbiology 65: 1288-1299 (2007)).
peable hilus nzae is an important and common respiratory pathogen
that causes otitis media in infants and children. NTHi is, after Streptococcus pneumoniae, the
most common cause of acute otitis media in children (J. Immunology 183: 2593-2601 (2009),
Pediatrics 113:1451-1465 (2004)). It is an important cause of sinusitis in children and adults.
(Current Infectious Disease Reports 11:177-182 (2009)). It has been associated with increased
risk of exacerbations in c obstructive pulmonary disease (COPD) in adults. (Journal of
Chronic Obstructive Pulmonary Disease 3:109-115 (2006)). In addition, non-typeable H.
influenzae causes community-acquired pneumonia in adults and may cause pneumonia in
children in developing countries. (Current Infectious Disease Reports 11:177-182 (2009)).
A need for vaccines for NTHi exists.
BRIEF SUMMARY OF THE INVENTION
As a first aspect, the present invention provides fusion proteins of a (I).
(X) m – (R1)n – A – (Y) o – B – (Z)p (formula I)
wherein:
X is a signal peptide or MHHHHHH (SEQ ID NO. 2);
m is 0 or 1;
R1 is an amino acid;
n is 0, 1, 2, 3, 4, 5 or 6;
A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, or PilA from
Haemophilus nzae or an immunogenic fragment thereof;
Y is selected from the group consisting of GG, SG, SS, GGG and (G)h wherein h is 4, 5, 6, 7, 8,
9, or 10;
o is 0 or 1;
B is PilA from Haemophilus influenzae or an immunogenic fragment f, or Protein E from
Haemophilus influenzae or an immunogenic nt thereof;
Z is GGHHHHHH (SEQ ID NO. 3); and
p is 0 or 1,
n when A is Protein E from hilus nzae or an immunogenic fragment thereof,
B is not Protein E from Haemophilus influenzae or an genic fragment thereof; and
wherein when A is PilA from Haemophilus influenzae or an immunogenic fragment thereof, B
is not PilA from hilus influenzae or an immunogenic fragment thereof.
As a second aspect, the present invention provides immunogenic compositions
comprising fusion proteins of formula (I). The composition may r comprise a
pharmaceutically acceptable adjuvant. The composition may comprise an excipient.
In a third aspect, the present invention provides a method for the treatment or prevention
of a condition or disease caused wholly or in part by Haemophilus influenzae. The method
comprises administering to a t in need thereof a therapeutically effective amount of the
fusion protein of formula (I). In a related aspect the invention provides for the use of a fusion
protein of formula (I) or an immunogenic composition thereof in the manufacture of a medicament
for the ent or prevention of a condition or disease caused wholly or in part by Haemophilus
influenza.
In a fourth aspect, the present invention provides a method for the treatment or prevention
of otitis media. The method comprises administering to a subject in need thereof a therapeutically
effective amount of the fusion protein of a (I). In a related aspect the invention provides for
the use of a fusion n of formula (I) or an immunogenic composition thereof in the
manufacture of a medicament for the treatment or prevention of otitis media
In a fifth aspect, the present invention provides a method for the treatment or prevention
of exacerbations in chronic obstructive pulmonary disease. The method comprises administering
to a subject in need thereof a therapeutically effective amount of the fusion protein of a (I).
In a related aspect the invention provides for the use of a fusion protein of formula (I) or an
immunogenic composition thereof in the cture of a medicament for the treatment or
prevention of exacerbations in chronic obstructive pulmonary disease.
(followed by 3A)
In a sixth aspect, the present invention provides a method for the treatment or prevention
of pneumonia. The method comprises administering to a t in need thereof a
therapeutically effective amount of the fusion n of formula (I). In a related aspect the
invention provides for the use of a fusion protein of formula (I) or an genic composition
thereof in the manufacture of a medicament for the treatment or prevention of nia.
In a seventh aspect, the present invention provides a pharmaceutical composition
comprising a fusion protein of formula (I) for use in the treatment or prevention of a condition or
disease caused wholly or in part by Haemophilus influenzae. Pharmaceutical compositions
may further comprise a pharmaceutically acceptable adjuvant.
In an eighth aspect, the present invention es nucleic acids encoding the proteins of
the invention.
In a ninth , the present invention provides a process of producing nucleic acids of
the invention.
Further aspects of the present invention are described in the detailed description of
particular embodiments, examples and claims which .
(followed by 4)
2012/050236
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. GE of induced bacterial extracts for fusion protein constructs LVL291, LVL268
and LVL269. Insoluble fraction (l), Soluble fraction (S) and Culture Media fraction (M) were
loaded for LVL291, LVL268 and LVL269 before and after ion (ind).
Figure 2. SDS—PAGE and Western blot related to purification extracts for fusion protein
ucts LVL291, LVL268 and LVL269. Flow through fraction (Ft), Wash fraction (W) and
Elution fraction (E) were loaded for purification of LVL291, LVL268 and LVL269. Anti-his tag
was used to probe extracts.
Figure 3. SDS-PAGE of induced bacterial and cation extracts for fusion protein constructs
LVL291 and LVL315. Culture Media on (M), Soluble fraction (Sol), lnsoluble fraction (lns),
Flow through fraction (Ft), Wash on #1 (W1), Wash fraction #2 (W2) and Elution fraction
(E) were loaded for LVL291 and LVL315.
Figure 4. SDS-PAGE of induced bacterial and purification extracts for fusion protein construct
LVL312. Culture Media fraction (M), Soluble fraction (Sol), ble fraction (lns), Flow Through
fraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E) were loaded
for LVL312.
Figure 5. SDS-PAGE of induced (1mM and 10uM IPTG) bacterial extracts for fusion protein
uct LVL317. Extracts from before (NI) and after induction (ln), Soluble fraction (S),
lnsoluble fraction (I).
Figure 6. SDS-PAGE of d (1mM and 10uM IPTG) bacterial extracts for fusion protein
construct LVL318. Extracts from before (NI) and after induction (ln), Culture Media fraction (M),
Soluble fraction (S), lnsoluble on (I).
Figure 7. CD spectra of PE, PilA and PE-PilA fusion proteins.
Figure 8. Combination of PE and PilA CD spectrum.
Figure 9. PilA thermal denaturation curve.
Figure 10. PE denaturation curve.
Figure 11. PE-PilA fusion protein l ration curve.
Figure 12. Typical SP SepharoseTM Fast Flow chromatogram.
Figure 13. Typical Q SepharoseTM Fast Flow chromatogram.
Figure 14. SDS-PAGE of ln-process samples from purification process of PE-PilA fusion
protein.
Figure 15. Western Blot of ln-process samples of purification process from PE-PilA fusion
protein. Blot using rabbit polyclonal anti-PE.
Figure 16. Western Blot of ln-process samples of cation process from PE-PilA fusion
protein. Blot using rabbit polyclonal anti-Eco/i (BLR).
Figure 17. Thermal transition of PE-PilA fusion protein and PE and PilA ns. Curves: PilA
(1), Protein E (Prot E, PE) (2), PE-PilA Purified Bulk not diluted, 737ug/ml (3), and PE-PilA
Purified Bulk d at Final Container tration 60ug/ml (4).
Figure 18. Antibody responses against LVL291 PE-PilA fusion n and against monovalent
PE and PilA in the Balb/c mouse model.
Figure 19. Effect of PE-PilA fusion protein vaccination on NTHi strain 86-028NP bacterial
clearance in mouse nasopharynx.
Figure 20. Effect of PE-PilA fusion protein ation on NTHi strain 3224A bacterial clearance
in mouse nasopharynx.
Figure 21. Effect of PilA vaccination on bacterial clearance in mouse nasopharynx.
2012/050236
Figure 22. Effect of PE vaccination on bacterial nce in mouse nasopharynx.
Figure 23. (a) LVL317 PE-PilA fusion protein binding to vitronectin and (b) LVL317 and LVL735
PE-PilA fusion protein bound to vitronectin.
Figure 24. tion of vitronectin binding by polyclonal antibodies against PE-PilA fusion
protein.
Figure 25. SDS-PAGE of e fractions of induced bacterial extracts for fusion protein
constructs LVL291, , LVL736, LVL737, LVL738, LVL739, LVL740 and pET26b vector
(negative control). (a) Experiment 1 (b) Experiment 2 (c) Experiment 3. PE-PilA fusion protein
indicated by arrow.
Figure 26. The average band percentage of fusion protein in the soluble fraction from
Experiments 1, 2 and 3.
Figure 27. PE and PilA antibody response to LVL317 and LVL735.
Figure 28. Effect of LVL735 and LVL317 vaccination on bacterial clearance in a mouse model
of non-typeable Haemophi/us influenzae nasopharyngeal zation.
DETAILED DESCRIPTION OF THE INVENTION
Unless othenNise explained or defined , all technical and scientific terms used
herein have the same g as commonly understood by one of ordinary skill in the art to
which this disclosure belongs. For example, definitions of common terms in molecular biology
can be found in Benjamin Lewin, Genes V, hed by Oxford University Press, 1994 (ISBN
0854287—9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 002182-9); and Robert A. Meyers (ed.), Molecular
Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH hers,
Inc., 1995 (ISBN 1569—8).
The singular terms “a, ” uan,” and “the” include plural referents unless context clearly
tes othenNise. Similarly, the word “or” is intended to include “and” unless the context
clearly indicates otherwise. It is r to be understood that all base sizes or amino acid
sizes, and all molecular weight or molecular mass values, given for nucleic acids or
polypeptides are approximate, and are provided for ption. Additionally, numerical
limitations given with respect to concentrations or levels of a substance, such as an n
may be approximate. Thus, where a concentration is indicated to be (for example)
approximately 200 pg, it is intended that the concentration includes values slightly more or
slightly less than (“about” or “~”) 200 pg.
Although methods and materials similar or equivalent to those described herein can be
used in the practice or g of this disclosure, le methods and materials are described
below.
The term “comprises” means “includes”. Thus, unless the context es othenNise,
the word “comprises,” and variations such as “comprise” and “comprising” will be understood
to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide,
antigen) or step, or group of compounds or steps, but not to the exclusion of any other
compounds, composition, steps, or groups thereof. The iation, “9.9.” is derived from
the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the
abbreviation “9.9.” is synonymous with the term “for example.”
In order to facilitate review of the various embodiments of this disclosure, the following
explanations of terms are ed. Additional terms and explanations are provided in the
context of this disclosure.
A “subject” as used herein is a mammal, including humans, non-human primates, and
non-primate s such as members of the rodent genus (including but not limited to mice
and rats) and members of the order Lagomorpha (including but not limited to rabbits).
As used herein in E”, “protein E”, “Prot E”, and “PE” mean Protein E from H.
influenzae. Protein E may consist of or comprise the amino acid sequence of SEQ ID NO. 4
(VKK T. GT.T.TACSAQI QKAEQNDVKL APPTDVRSGY IIRLVKNVNYY
DSfiS WVDW QfiL’Q VHE'DA WNLDKGLYV YPEPKRYARS LNCA
NY {LTQVQTD FY33 FWGQGL RAAP {KQKKH TLSLTPDTTL YWAAQ CAN
YGEAFSVDKQ as well as sequences with at least or exactly 75%, 77%, 80%, 85%, 90%,
95%, 97%, 99% or 100% identity, over the entire length, to SEQ ID NO. 4. Comparison of 53
sequences of Protein E from Haemophi/us influenzae (Table 1, SEQ ID NO. 5 — SEQ ID NO.
57) demonstrated approximately 77% to approximately 100% identity to n E as set forth
in SEQ ID NO. 4. For example, in the amino acid sequence of Protein E, amino acid #20 may
be isoleucine (I) or threonine (T); amino acid #23 may be alanine (A) or valine (V); amino acid
#24 may be lysine (K) or glutamic acid (E); amino acid #31 may be alanine (A) or threonine
(T); amino acid #32 may be e (P) or alanine (A); amino acid #34 may be threonine (T) or
alanine (A); amino acid #37 may be arginine (R) or glutamine (Q); amino acid #47 may be
valine (V) or alanine (A); amino acid #57 may be tryptophane (W) or may be absent (-); amino
acid #70 may be e (A) or threonine (T); amino acid #93 may be glutamine (Q) or absent
(-); amino acid #109 may be threonine (T) or isoleucine (I); amino acid #119 may be glycine
(G) or serine (S); amino acid #153 may be glutamic acid (E) or lysine (K); amino acid #156
may be serine (S) or leucine (L); amino acid #160 may be lysine (K) or gine (N); amino
acid #161 may be lysine (K), isoleucine (I) or absent (-); amino acids #162 - #195 may be
absent, or as set forth in SEQ ID NO. 15 (with (-) indicating amino acid #166 is absent) or as
set forth in SEQ ID NO. 16; or any combination thereof.
Protein E may consist of or se an amino acid sequence that differs from SEQ ID
NO. 4 at any one or more amino acid selected from the group consisting of: amino acid #20,
amino acid #23, amino acid #24, amino acid #31, amino acid #32, amino acid #34, amino acid
#37, amino acid #47, amino acid #57, amino acid #70, amino acid #93, amino acid #109,
amino acid #119, amino acid #153, amino acid #156, amino acid #160, amino acid #161 and
amino acids #162-#195, wherein amino acid #20 is threonine (T); amino acid #23 is valine (V);
amino acid #24 is lysine (K); amino acid #31 is threonine (T); amino acid #32 is alanine (A);
amino acid #34 is alanine (A); amino acid #37 is glutamine (Q); amino acid #47 is e (A);
amino acid #57 is absent (-); amino acid #70 is threonine (T); amino acid #93 is absent (-);
amino acid #109 is isoleucine (I); amino acid #119 is serine (S); amino acid #153 is lysine (K);
amino acid #156 is leucine (L); amino acid #160 is asparagine (N); amino acid #161 is lysine
(K) or isoleucine (I); or amino acids #162 - #195 are as set forth in SEQ ID NO. 15 (with (-)
indicating amino acid #166 is absent) or as set forth in SEQ ID NO. 16.
Table 1: Protein E amino acid sequences from 53 strains of Haemophi/us influenzae
(SEQ ID NO. 5 - SEQ ID NO. 57). - indicates amino acid is absent.
m Protein E ce
.G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.5)
RdKWZO .G.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.DQG.YVYPEPKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK
_ .YNAAQIICAVYGKAFSVDK< SEQ ID V0.6)
86-028NP CSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
_ .YNAAQIICAVYGEAFSVDK< SEQ ID V0.7)
R2846 .G.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
_ 1T.YNAAQIICA_VYGKAFSV < SEQ ID V0.8)
R2866 .G.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
_ 1T.YNAAQIICA_VYGKAFSV < SEQ ID V0.9)
3655 .G.L"ACSAQIQ{AEQNDMK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
_ 1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.10)
PittAA .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
_ 1T.YNAAQIICA_VYGEAFSV < SEQ ID V0.11)
.G.L"ACSAQIQ{AEQNDMK.A?P"DVRSGYIQLVKNVNYYIDSESI-VDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGKAFSV {(SEQ ID N0.12)
.G.L"ACSAQIQ{AEQNDVK.APPTDVRSGYIRLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGKAFSVDK< (SEQ ID N0.13)
.L"ACSAQIQKAEQNDVK.APP"DVRSGYIRLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICANYGKAFSVDKK SEQ ID No.14)
.L"ACSAQTQKAEQNDVK.TPP"DVQSGYVRLVKNVNYYIDSESIWVDNQ
PEPKRYARSVRQYKILNCANYHLTQVRIDFYDEFWGQGLRAAPK
?Dr .YNAAQIICANYGKAFSVDKNKKICT—LISLNFIQLLGCREYSIFLQLLLFYC
EQ ID No.15)
.L"ACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
"PDF .YNAAQIICANYGKAFSVDKKIKKICTLISLNFIQLLGCREYSIFLQLLLFYC
EQ ID No.16)
.G.L"ACSAQIQ{AEQNDMK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ
VYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICANYGKAFSV < SEQ ID No.17)
.G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICANYGKAFSVDK< SEQ ID No.18)
.G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_NYGEAFSVDK< SEQ ID No.19)
038144S1 CSAQTQ<VEQNDVK.TAP"DVRSGFVQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_NYGKAFLVDK< SEQ ID No.20)
810956 .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_NYGEAFSV < SEQ ID No.21)
821246 .G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_NYGKAFSVDK< SEQ ID No.22)
840645 .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.23)
902550Zl9 .L"ACSAQTQ<VEQNDVK.T?P"DVRSGYVQLVKNVNYYIDSESIWVDNQ
PEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.24)
A840l77 .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.25)
A860514 .L"ACSAQTQ<VEQNDVK.TAP"DVRSGYVQLVKNANYYIDSESIWVDNQ
PEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.26)
A950014 .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRIDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.27)
306543X4 .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK<(SEQ ID N0.28)
A930105 AQIQ{AEQNDVK.A?PTDVRSGYIRLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK<(SEQ ID N0.29)
901905U AQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.30)
A920030 .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.31)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
IVHFDAVVV {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSV
27Wll679lN CSAQTQ<VEQNDVK.T?P"DVRSGYVQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGKAFSV < SEQ ID V0.33)
.G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.34)
.G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGEAFSVDK< SEQ ID V0.35)
.G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGEAFSVDK< SEQ ID V0.36)
.G.L"ACSAQTQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ
VYPEPKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.37)
.G.L"ACSAQTQ<VEQNDVK.TAPADVRSGYVQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
AQIICA_VYGKAFSVDK< SEQ ID V0.38)
.G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.39)
.G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYAQSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.40)
.G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.41)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
IICAVYGKAFSVDK< SEQ ID V0.42)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.43)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.44)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQSLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.45)
.T."‘ACSAQTQ {VEQNDVK .T E’Pr1DVRSGYVQLVKNVNYYI DSES IWVDNQI
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
1TVYNAAQIICAVYGKAFSVDK< SEQ ID V0.46)
.GT.T."ACSAQTQ {VEQNDVK .T SGYV QLVKNVNYYI DSES IWVDNQI
.DKG.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
AQIICAVYGKAFSVDK< SEQ ID V0.47)
.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.48)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.49)
.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
{G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK<(SEQ ID N0.50)
{KII .L"ACSAQTQ{AEQNDVK.TPPTDVRSGYIRLVKNVNYYIDSESIWVDNQ
IVHFDAVVV {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSV
.."ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.52)
AQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.53)
. ."ACSAQTQ VK .A .3?" DVRSGYI QLVKNVNYYI DSES IWVDNQI
PKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK<(SEQ ID N0.54)
. ."ACSAQIQ {AKQNDVKEA .3?" DVRSGYI QLVKNVNYYI DSES I
.YVYPEPKRYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGEAFSVDK< SEQ ID V0.55)
.."ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ
.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.56)
.."ACSAQIQ{AEQNDMK.A?P"DVRSGYIQLVKNVNYYIDSESI-VDNQ
.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK
.YNAAQIICAVYGKAFSVDK< SEQ ID V0.57)
n E may be Protein E from H. influenzae strain 3224A, RdKW20, 86-028NP,
R2846, R2866, 3655, PittAA, PittEE, PittHH, PittII, R3021, 22.4-21, 3219C, 3185, 3241A,
03814481, 810956, 821246, 840645, 902550Z19, A840177, A860514, A950014, 306543X4,
A930105, U, A920030, 3221B, 27W116791N, N218, N163, N162, N107, N91,
D211PG, D211PD, , D201PD, D198PG, D198PD, D195PD, D189PG, D189PD,
D129CG, D124PG, D124PD, D58PG, D33OD, B8433, B8432, 1714, 1128 or B8430. Protein
E may be Protein E as set forth in any of SEQ ID NO. 5 — SEQ ID NO. 57.
Protein E may be a sequence with at least 95% identity, over the entire length, to any of
SEQ ID NO. 4 — SEQ ID NO. 57. Protein E may be a sequence with at least 95% identity,
over the entire length, to any of the sequences set forth in Table 1, SEQ ID NO. 5 — SEQ ID
NO. 57.
Immunogenic fragments of Protein E comprise immunogenic fragments of at least 7, 10,
, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 4. The immunogenic nts
may elicit antibodies which can bind SEQ ID NO. 4.
Immunogenic nts of Protein E may comprise immunogenic fragments of at least
7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of any of SEQ ID NO. 4 — SEQ ID NO. 57.
The immunogenic fragments may elicit antibodies which can bind the full length sequence
from which the fragment is derived.
Immunogenic fragments of Protein E comprise immunogenic fragments of at least 7, 10,
, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 5 — SEQ ID NO. 57. The
immunogenic fragments may elicit antibodies which can bind the full length sequence from
which the fragment is derived.
As used herein “PilA” means PiIin A from H. influenzae. PilA may consist of or comprise
the protein sequence of SEQ ID NO. 58 (MKLTTQQTLK KGh'TT. *ZTM V A A TAT
A BSYQNYT ELLQ ASAPYKADVIfiL‘L LCVYSTNIZTT.L NCTGGKNGIA
ADITTAKGYV KSVTTSWGA: TVKGDGTLAN M*'.Y T.QATGN AATGVTWTTT
SLFP VTQ) as well as sequences with 80% to 100% identity to SEQ ID
NO. 58. For example, PilA may be at least 80%, 85%, 90%, 95%, 97% or 100% identical to
SEQ ID NO. 58. Full length comparison of 64 ces of PilA from hi/us
influenzae (Table 2, SEQ ID NO. 58 — SEQ ID NO. 121) demonstrated approximately 80% to
100% ty to PilA as set forth in SEQ ID NO. 58. For example, in the amino acid
sequence of PilA, amino acid #6 may be glutamine (Q) or leucine (L); amino acid #7 may be
glutamine (Q) or threonine (T); amino acid #37 may be ine (Q) or lysine (K); amino acid
#44 may be alanine (A) or serine (S); amino acid #57 may be alanine (A) or serine (S); amino
acid #67 may be asparagine (N) or glycine (G); amino acid #68 may be glutamic acid (E) or
lysine (K); amino acid #69 may be theronine (T) or proline (P); amino acid #71 may be lysine
(K), asparagine (N), serine (S) or threonine (T); amino acid #73 may be threonine (T), serine
(S) or methionine (M); amino acid #76 may be lysine (K), serine (S) or asparagine (N); amino
acid #84 may be threonine (T) or lysine (K); amino acid #86 may be alanine (A) or valine (V);
amino acid #91 may be lysine (K) or alanine (A); amino acid #94 may be threonine (T),
isoleucine (I) or lysine (K); amino acid #96 may be serine (S) or glutamine (Q); amino acid #97
may be asparagine (N) or serine (8); amino acid #99 may be alanine (A) or glycine (G); amino
acid #103 may be alanine (A) or lysine (K); amino acid #109 may be aspartic acid (D), alanine
(A) or threonine (T); amino acid #110 may be glycine (G), asparagine (N), or arginine (R);
amino acid #112 may be serine (S) or glutamic acid (E); amino acid #114 may be threonine
(T) or isoleucine (l); amino acid #116 may be threonine (T) or glutamine (Q); amino acid #118
may be glutamic acid (E), threonine (T), alanine (A), lysine (K) or serine (8); amino acid #121
may be serine (S) or alanine (A); amino acid #122 may be alanine (A) or threonine (T); amino
acid #123 may be lysine (K), threonine (T) or alanine (A); amino acid #128 may be lysine (K)
or threonine (T); amino acid #135 may be aspartic acid (D) or glutamic acid (E); amino acid
#136 may be alanine (A) or threonine (T); amino acid #145 may be glycine (G) or arginine (R);
amino acid #149 may be glutamine (Q) or lysine (K); or any ation thereof.
Pil A may consist of or se an amino acid sequence that differs from SEQ ID NO.
58 at any or more amino acid selected from the group consisting of amino acid #6, amino acid
#7, amino acid #37, amino acid #44, amino acid #57, amino acid #67, amino acid #68, amino
acid #69, amino acid #71, amino acid #73, amino acid #76, amino acid #84, amino acid #86,
amino acid #91, amino acid #94, amino acid #96, amino acid #97, amino acid #99, amino acid
#103, amino acid #109, amino acid #110, amino acid #112, amino acid #114, amino acid
#116, amino acid #118 amino acid, #121, amino acid #122, amino acid #123, amino acid
#128, amino acid #135, amino acid #136, amino acid #145 and amino acid #149, wherein
amino acid #6 is leucine (L); amino acid #7 is threonine (T); amino acid #37 is lysine (K);
amino acid #44 is serine (8); amino acid #57 is serine (8); amino acid #67 is glycine (G);
amino acid #68 is lysine (K); amino acid #69 is proline (P); amino acid #71 is lysine (K), serine
(S) or threonine (T); amino acid #73 is serine (S) or methionine (M); amino acid #76 is serine
(S) or asparagine (N); amino acid #84 is lysine (K); amino acid #86 is valine (V); amino acid
#91 is e (A); amino acid #94 is isoleucine (l) or lysine (K); amino acid #96 is glutamine
(Q); amino acid #97 is serine (8); amino acid #99 is glycine (G); amino acid #103 is alanine
(A); amino acid #109 is aspartic acid (D) or threonine (T); amino acid #110 is e (G) or
arginine (R); amino acid #112 is serine (8); amino acid #114 is threonine (T); amino acid #116
is threonine (T); amino acid #118 is ic acid (E), alanine (A), lysine (K) or serine (8);
amino acid #121 is serine (8); amino acid #122 is threonine (T); amino acid #123 is lysine (K)
or alanine (A); amino acid #128 is lysine (K); amino acid #135 is ic acid (E); amino acid
#136 is threonine (T); amino acid #145 is arginine (R); amino acid #149 is lysine (K).
Table 2: Pilin A amino acid sequences from 64 strains of Haemophi/us influenzae (SEQ
ID NO. 58—SEQ ID NO. 121).
m PilA sequence
86-028NP 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.58)
NTHi3219C 1QQTI{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKADVELCVY
I KNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGVISYTLTAEGDSAKGVTWK
1DASEFPANFCGSV"Q (SEQ ID No.59)
24A 1QQTI{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
I 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV'EQ (SEQ ID No.60)
NTHllZ MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSSCSGGSNGIAADI"TAKGYVASVITQSGGITVKGDGTLANWEYILQAAGNAAAGVTWT
T"CKGTDASEFPANFCGSV"Q (SEQ ID No.61)
1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.62)
NTHi67 MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKSDVELCVY
S"GKPSTCSGGSNGIAADI"TVKGYVKSV"TSNGAITVAGNGTLDGWSYTLTAEGDSAKGVTWT
DASEFPANFCGSV"Q (SEQ ID No.63)
1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.64)
1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV'DQ (SEQ ID No.65)
MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVZ
1NCTGGKNGIAADI"TAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWT
1DASEFPANFCGSV'EQ (SEQ ID No.66)
QTL{KGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSE.LQASAPYKADVELCVY
S"NEI"NCMGGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAAAGVTWT
DASEFPANFCGSI"Q (SEQ ID No.67)
1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANVIEYILQAKGNATAGVTWT
1DASEFPANFCQSV"K (SEQ ID No.68)
.QT.{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1SC"GGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANVIEYILQAKGNAAAGVTWT
1DASEFPANFCGSVTK (SEQ ID No.69)
MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSSCSGGSNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANWEYILQASGNAATGVTWT
T"CKG"DASEFPANFCGSV"Q (SEQ ID No.70)
.QT.{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1SC"GGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANVIEYILQAKGNAAAGVTWT
1DASEFPANFCGSVTK (SEQ ID No.71)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQATGNAATGVTWT
T"CKG"DASEFPANFCGSV"Q (SEQ ID No.72)
MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKSDVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT
T"CKGTDASEFPANFCQSV"K (SEQ ID No.73)
KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.74)
1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1EAS.FPANFCGSV"Q (SEQ ID No.75)
1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKADVELCVY
KNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANVIEYILQAKGNATAGVTWT
1DASEFPANFCQSV"K (SEQ ID No.76)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQATGNAATGVTWT
T"CKG"DASEFPANFCGSV"Q (SEQ ID No.77)
MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT
T"CKGTDASEFPANFCQSV"K (SEQ ID No.78)
1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1EAS.FPANFCGSV"Q (SEQ ID No.79)
1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
GKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1EAS.FPANFCGSV"Q (SEQ ID No.80)
MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKSDVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGWSYTLTAEGDSAKGVTWK
T"CKGTDASEFPANFCGSV"K (SEQ ID No.81)
MKLTTQQT.{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"NI KNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT
T"C 1DASEFPANFCGSV"Q (SEQ ID No.82)
MKLTTQQT.{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"NEA"KC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANWEYILQASGNAATGVTWT
T""C 1DASEFPANFCGSV'EQ (SEQ ID No.83)
1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.84)
038144Sl MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAISE.LQASAPYKSDVELCVY
STGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT
TTCKGTDAS. EQ ID No.85)
821246 LIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1EAS.FPANFCGSV"Q (SEQ ID No.86)
840645 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.87)
Zl9 MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKSDVELCVY
S"GKPSTCSGGSNGIAADI"TVKGYVKSV"TSNGAITVAGNGTLDGWSYTLTAEGDSAKGVTWT
T"CKGTDASEFPANFCGSV"Q (SEQ ID No.88)
A840177 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
KNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV'EQ (SEQ ID No.89)
A920030 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1NC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV'EQ (SEQ ID No.90)
A950014 MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKADVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDRMSYTLTAEGDSAKGVTWT
T"CKGTDASEFPANFCGSV"Q (SEQ ID No.91)
90l905U MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSSCSGGSNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANWEYILQASGNAATGVTWT
T"CKGTDASEFPANFCGSV"Q (SEQ ID No.92)
A920029 1QTT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKSDVELCVY
1NC"GGKNGIAADI"TAKGYVASVITQSGGITVKGNGTLTNVIEYILQATGNAATGVTWT
1DASEFPANFCGSI"Q (SEQ ID No.93)
A930105 .QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1CKG"DASEFPANFCGSVTQ (SEQ ID No.94)
306543X4 MKLTTQQTL<KGFTLIELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY
S"GKPSSCSGGSNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT
T"CKGTDASEFPANFCGSV"Q (SEQ ID No.95)
MKLTTQQF.<KGFTLIELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY
S"NI "KC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT
Tmc 1Dms CGSV"Q (SEQ ID No.96)
1QQr .(KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY
"NC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.97)
1QQT.<KGFTT.IELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY
"NC"GGKNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (SEQ ID No.98)
MKLTTQQTL<KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQAKGNATAGVTWT
T"CKGTDASEFPANFC QSV"K (SEQ ID No.99)
MKLTTQQTL<KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQAKGNATAGVTWT
T"CKGTDASEFPANFC QSV"K (SEQ ID No.100)
"QQT - {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'.
4 .LQASAPYKADVELCVY
"NC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DAS EFPANFCGSV"Q (SEQ ID )
MKLTTQQTL<KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQAKGNATAGVTWT
T"CKGTDASEFPANFC QSV"K (SEQ ID No.102)
MKLTTQQT.<KGFTLIELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY
S"NI "KC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT
T""C 1DAS CGSV"Q (SEQ ID No.103)
1QQT.<KGFTT.IELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 PYKADVELCVY
"TNC"GGKNGIAADITTAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
TTCKGTDAS.4FPANFCGSVTQ (SEQ ID NO.IO4)
"QQT. {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY
"NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (S EQ ID No.105)
"QQT _‘
- {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'. .LQASAPYKADVELCVY
"NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIIEYILXATGNAATGVTWT
1DASEFPANFCGSV"Q (S EQ ID NO.IO6)
"QQT - {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'.
4 .LQASAPYKADVELCVY
"NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (S EQ ID NO.IO7)
"QQT _‘
- {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'. .LQASAPYKADVELCVY
"NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT
1DASEFPANFCGSV"Q (S EQ ID NO.IO8)
MKLTT.QTL {KGFTTI'ELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY
S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT
T"CKGF1DASEFPANFCGSVTQ (S EQ ID NO.IO9)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE PYKADVELCVY
S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT
T"CKG"DASEFPANFCGSVTQ (SEQ ID NO.llO)
1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASAE’YKADVELCVY
1SC"GGKNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGVISYTLTAEGDSAKGVTWK
1DAS.4FPANFCGS\/""Q (SEQ ID NO.lll)
KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASAE’YKADVELCVY
1SC"GGKNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGVISYTLTAEGDSAKGVTWK
1DASEFPANFCGSV"Q (SEQ ID No.112)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE .LQASAPYKADVELCVY
S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT
DASEFPANFCGSVTQ (SEQ ID NO.ll3)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT
T"CKG"DASEFPANFCGSVTQ (SEQ ID NO.ll4)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT
Tr 1DASEFPANFCGSVTQ (SEQ ID No.115)
1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1EAS.FPANFCGSV"Q (SEQ ID NO.ll6)
.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
1GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT
1CKG"DAS_JFPANFCGSVTQ (SEQ ID )
MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT
T"CKGTDASEFPANFCQSV"K (SEQ ID NO.ll8)
MKLTTQQT.{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"NI KNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT
T""C 1DASEFPANFCGSV'EQ (SEQ ID NO.ll9)
MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQATGNAATGVTWT
T'ECKG'EDASEFPANFCGSV'EQ (SEQ ID No.120)
MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKSDVELCVY
S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT
T"CKGTDASEFPANFCQSV"K (SEQ ID No.121)
PiIA may be PiIA from H. influenzae strain NTHi3219C, NTHi3224A, NTHi12, NTHi44,
NTHi67, 1054MEE, E, 1728MEE, E, 1060MEE, RdKW20, 214NP, 1236MEE,
1714MEE, E, 86-028NP, R2846, R2866, 3655, PittAA, PittGG, , R3021, 22.4-21,
3185A, 3221B, 3241A, 03814481, 821246, 840645, 902550Z19, A840177, A920030,
4, 901905U, A920029, A930105, 306543X4, N218, N163, N162, N120, N107, N92,
N91, D219PG, D211PG, D211PD, D204CD, D198PG, D198PD, D195PD, D195CD, D189PG,
D189PD, D124PG, D124PD, D124CG, D58PG, BS433, BS432, BS430, 1714 or 1128. An
amino acid sequence for PilA from H. influenzae strain D204CD is set forth in SEQ ID NO.
106, wherein X at position #116 is either glutamine (Q) or Ieucine (L); ambiguity as to the
amino acid at position #116 could be cleared up by cal tion of the second
nucleotide encoding amino acid #116, clarifying the PilA sequence for strain D204CD. PilA
may be PilA as set forth in any of SEQ ID NO. 58 — SEQ ID NO. 121.
PilA may be a sequence with at least 95% identity, over the entire length, to any of SEQ
ID NO. 58 — SEQ ID NO. 121 (as set out in Table 2).
lmmunogenic fragments of PilA comprise immunogenic fragments of at least 7, 10, 15,
, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58 — SEQ ID NO. 121. The
genic fragments may elicit dies which can bind the full length sequence from
which the fragment is derived.
For example, genic nts of PilA comprise immunogenic fragments of at
least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58. The immunogenic
nts may elicit antibodies which can bind SEQ ID NO. 58.
Identity between polypeptides may be calculated by various algorithms. For example,
the Needle program, from the EMBOSS package (Free software; : The European
Molecular Biology Open Software Suite (2000). Trends in Genetics 16(6): 276—277) and the
Gap program from the GCG® package (Accelrys Inc.) may be used. This Gap program is an
implementation of the Needleman-Wunsch algorithm described in: Needleman, S. B. and
Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The BLOSUM62 scoring matrix has been
used, and the gap open and extension penalties were respectively 8 and 2.
Looking at the computed alignment, identical residues between two compared
sequences can be observed. A percentage of identity can be computed by (1) ating
the number of identities divided by the length of the alignment, multiplied by 100 (for example,
for the Needle program analysis), (2) calculating the number of identities divided by the length
of the longest sequence, multiplied by 100, (3) calculating the number of ties divided by
the length of the st sequence, multiplied by 100, or (4) calculating the number of
identities divided by the number of aligned residues, multiplied by 100 (a residue is aligned if it
is in front of another) (for e, for the Gap program analysis).
As used herein, “adjuvant” means a nd or substance that, when administered to
a subject in conjunction with a vaccine, immunotherapeutic, or other antigen- or gen-
containing composition, increases or enhances the subject’s immune response to the
administered antigen or immunogen (as compared to the immune response that would be
obtained in the absence of adjuvant). This is to be guished from “adjuvant therapy”,
defined by the National Cancer Institute of the United States Institutes of Health in the context
of cancer treatment as additional treatment given after the primary treatment, to lower the risk
that the cancer will recur.
Conservative substitutions are well known and are generally set up as the default
scoring matrices in sequence ent computer programs. These programs include
PAM250 (Dayhoft MO. et al., , “A model of evolutionary changes in proteins”, In “Atlas
of Protein sequence and structure” 5(3) MO. Dayhoft (ed.), 345-352), National Biomedical
Research Foundation, gton, and Blosum 62 (Steven Henikoft and Jorja G. Henikoft
(1992), “Amino acid substitution matrices from protein blocks”), Proc. Natl. Acad. Sci. USA 89
(Biochemistry): 10919. The invention further provides fusion proteins of formula (I)
containing vative amino acid substitutions. For example, the fusion proteins of formula
(I) may contain a vative substitution of any amino acid from PE or PilA of H. influenzae
as described in any of the sequences set forth herein (for example, any PE sequence set forth
in SEQ ID NO. 4 — SEQ ID NO. 57 and/or any PilA sequence set forth in SEQ ID NO. 58 —
SEQ ID NO. 121)
As used herein “signal peptide” refers to a short (less than 60 amino acids, for e,
3 to 60 amino acids) polypeptide present on precursor proteins (typically at the N terminus),
and which is typically absent from the mature protein. The signal peptide (sp) is typically rich
in hydrophobic amino acids. The signal peptide directs the transport and/or secretion of the
translated protein h the membrane. Signal peptides may also be called targeting
signals, transit peptides, localization signals, or signal sequences. For example, the signal
sequence may be a co-translational or post-translational signal peptide.
A heterologous signal peptide may be cleaved from a fusion protein construct by signal
peptide ases during or after protein transportation or ion. For example, the signal
peptide peptidase is signal peptide peptidase l. A “heterologous” signal peptide is one which
is not ated with the protein as it exists in nature.
As used herein “treatment” means the prevention of occurrence of symptoms of the
condition or disease in a subject, the prevention of recurrence of symptoms of the condition or
disease in a subject, the delay of recurrence of symptoms of the condition or e in a
subject, the decrease in severity or frequency of symptoms of the condition or disease in a
subject, slowing or eliminating the progression of the condition and the partial or total
elimination of symptoms of the disease or condition in a subject.
As used herein, “optionally” means that the subsequently described s) may or may
not occur, and includes both event(s) that occur and events that do not occur.
The pathogenesis of disease caused by NTHi begins with nasopharyngeal colonization.
Mechanisms to adhere to and maintain erm residence within the nasopharyngeal micro-
environment are considered ‘virulence determinants’ for NTHi. (Vaccine 28: 279-289 (2010)).
The importance of NTHi being able to adhere to the mucosal epithelial surfaces of a
human host is reflected in the multiplicity of adhesins expressed by NTHi. For example,
some NTHi express pili. Other adhesive structures belong to the autotransporter family of
proteins; these e Hap, HMW1/HMW2 and Hia/Hsf proteins. Further outer membrane
proteins, such as the P2 protein, P5 protein and OapA have been described as adhesions for
Haemophi/us influenzae. (Cellular Microbiology 4:191-200 , Microbes and Infection 10:
87-96 (2008), e 28: 279-289 ).
Otitis media is a major cause of morbidity in 80% of all children less than 3 years of age.
(Expert Rev. Vaccines 5:517-534 (2006)). More than 90% of children develop otitis media
before age 7 (Current Opinion in lnvestigational Drugs 4:953-958 ). In 2000, there were
16 million visits made to -based ians for otitis media in the United States and
approximately 13 million antibacterial prescriptions dispensed. (Pediatrics 113:1451-1465
(2004)). In European countries, the reported acute otitis media rates range between 0.125 to
1.24 per year. (Expert Review of es 81479-1500 (2009)). Otitis media is a costly
infection and the most common reason children receive antibiotics. (Current ious
Disease Reports —182 (2009)). Bacteria are responsible for imately 70% of
cases of acute otitis media, with Streptococcus pneumoniae, non-typeable Haemophi/us
influenzae, and Moraxe/Ia catarrha/is predominating as the causative agents (Expert Review
of Vaccines 5:517-534 (2006)). A subset of children experience recurrent and chronic otitis
media and these otitis prone children have protracted middle-ear effusions that are associated
with hearing loss and delays in speech and ge development. (Current Infectious
e Reports 11:177-182 (2009)).
ing the introduction of the heptavalent pneumococcal vaccine in many countries,
some studies have demonstrated a significant increase in the proportion of acute otitis media
caused by H. zae, with H. influenzae becoming the predominant pathogen. (Pediatric
Infectious e Journal -828; Pediatric ious Disease Journal -833
(2004)).
Since otitis media is a multifactorial disease, the feasibility of preventing otitis media
using a vaccination strategy has been questioned. (Current Infectious Disease Reports
11:177-182 (2009)). However, the s from one study suggest that it is possible for an
n to induce at least partial protection t non-typeable H. influenzae. (Lancet
367:740—748 (2006)). One approach to developing vaccine antigens is to use antigenically
conserved regions of genetically heterogeneous but abundantly expressed surface molecules.
Another approach is to identify surface proteins that demonstrate sequence or functional
epitope conservation. A third consideration for a vaccine antigen could be to select an antigen
that is expressed during infection and colonization in a human host. Murphy (Curr. Infect.
e Reports 11:177-182 (2009) states that, despite the existence of several potential non-
typeable H. influenzae candidate antigens, one cannot predict with certainty whether the
candidate antigen will be ive. (Current Infectious Disease Reports 11:177-182 (2009)).
Some of the proteins bed as potential vaccine antigens are: Haemophi/us adhesin
protein (Hap), High molecular-weight (HMVV) proteins 1 and 2, H. influnzae adhesin (Hia), D15
protein, HtrA heat shock protein, P2 surface protein, Iipoprotein D, P5 fimbrin derived
peptides, outer membrane protein P4, outer membrane protein (OMP) 26 (OMP26), P6
protein, Protein E, Type IV pilus, Iipooligosaccharide and phosphoryl e. (Current
Infectious e Reports 11:177-182 (2009); Expert Review of Vaccines 5:517—534 (2006)).
The chinchilla model is a robust and validated animal model of otitis media and its
prevention (Expert Review of Vaccines 8:1063-1082 (2009)). While the illa model may
mimic the natural course of human infection, others have suggested that results in the
chinchilla model may vary from one laboratory to the next. (Current Opinion in Investigational
Drugs 4:953-958 (2003)).
WO 39225
Various other rodents have also been used for the induction of otitis media and are
summarized in Vaccine 26:1501-1524 (2008). The murine animal model is often d in
otitis media research.
The presence of bactericidal antibody is associated with protection from otitis media due
to non-typeable H. influenzae. (Current n in ious e -134 (2003)).
However, an immune response need not be bactericidal to be effective against NTHi.
Antibodies that merely react with NTHi surface adhesins can reduce or eliminate otitis media
in the chinchilla. (Current Opinion in lnvestigational Drugs 4:953-958 (2003)).
Chronic obstructive pulmonary disease is a chronic inflammatory disease of the lungs
and a major cause of morbidity and mortality worldwide. imately one in 20 deaths in
2005 in the US had COPD as the underlying cause. (Drugs and Aging 26:985-999 (2009)). It
is projected that in 2020 COPD will rise to the fifth g cause of disability adjusted life
years, chronic invalidating es, and to the third most important cause of mortality (Lancet
349:1498-1504 (1997)).
The course of COPD is characterized by progressive worsening of airflow limitation and
a decline in pulmonary function. COPD may be complicated by frequent and recurrent acute
exacerbations (AE), which are associated with enormous health care expenditure and high
morbidity. (Proceedings of the American Thoracic Society 4:554—564 (2007)). One study
suggests that approximately 50% of acute exacerbations of ms in COPD are caused
by non-typeable Haemophi/us influenzae, /Ia catarrha/is, Streptococcus pneumoniae,
and Pseudomonas nosa. (Drugs and Aging 26:985-999 (2009)). H. influenzae is found
in 20-30% of exacerbations of COPD; Streptococcus pneumoniae, in 10-15% of
exacerbations of COPD; and Moraxe/la catarrha/is, in 10-15% of exacerbations of COPD.
(New England l of Medicine 359:2355-2365 (2008)). Haemophi/us influenzae,
Streptococcus pneumoniae, and Moraxe/Ia catarrha/is have been shown to be the primary
pathogens in acute exacerbations of bronchitis in Hong Kong, South Korea, and the
Phillipines, while K/ebsie/la spp., Pseudomonas aeruginosa and Acinetobacter spp. constitute
a large proportion of pathogens in other Asian countries/regions including lndonesia, Thailand,
Malaysia and Taiwan (Respirology, (2011) 16, 532-539;
doi:10.1111/j.1440.1843.2011.01943.x). ln Bangladesh, 20% of patients with COPD showed
positive sputum e for Pseudomonas, K/ebsie/Ia, Streptococcus niae and
Haemophi/us influenzae, while 65% of patients with AECOPD showed positive cultures for
Pseudomonas, K/ebsie/Ia, Acinetobacter, Enterobacter, Moraxe/Ia catarrha/is and
combinations thereof. (Mymensingh Medical Journal 19:576-585 (2010)). However, it has
been ted that the two most important measures to prevent COPD exacerbation are
active immunizations and chronic maintenance of cotherapy. (Proceedings of the
American Thoracic Society 4:554—564 (2007)).
There is a need for effective vaccines against NTHi. Using antigens that may act at
different steps in pathogenesis may improve the efficacy of a vaccine. The inventors have
found that PilA and PE may be beneficially present in the immunogenic compositions of the
invention as fusion ns.
The present invention relates to fusion proteins of formula (I).
(X) m - (R1)n — A — (Y) o — B — (Z)p (formula I)
wherein:
X is a signal peptide or MHHHHHH (SEQ ID NO. 2);
m is 0 or 1;
R1 is an amino acid;
n is 0, 1, 2, 3, 4, 5 or6;
A is Protein E from Haemophi/us influenzae or an immunogenic nt thereof, or PilA from
Haemophi/us zae or an genic fragment thereof;
Y is selected from the group consisting of GG, SG, 88 and (G),1 wherein h is 4, 5, 6, 7, 8, 9, or
o is 0 or 1;
B is PilA from Haemophi/us influenzae or an immunogenic fragment thereof, or Protein E from
Haemophi/us influenzae or an immunogenic fragment thereof;
Z is HH (SEQ ID NO: 3); and
p is 0 or 1.
In one embodiment, the fusion proteins of formula (I) are defined wherein X is selected
from the group ting of the signal sequence from CcmH hrome c membrane
n H), DsbA (periplasmic protein disulfide isomerise l), DsbB (disulfide bond membrane
protein B), FlgI (flagellar peptidoglycan ring protein), FocC (F1c Chaperone protein), MalE
(maltose transporter t E), NadA (quinolinate synthase subunit A), NikA (nickel ABC
transporter component A), NspA (Neisserial surface protein A), Omp26 (outer membrane
protein 26), OmpA (outer ne protein A), OspA (outer surface n A), pelB (pectate
lyase B), PhoA (bacterial alkaline phosphatase), PhtD (pneumococcal histidine triad protein
D), PhtE ococcal histidine triad protein E), SfmC (periiplasmic pilin chaperone), Sip1
(surface immunogenic protein), TolB (Tol-Pal Cell Envelope x Component B), TorA
(trimethylamine e reductase system subunit A), TorT (trimethylamine N-oxide reductase
system periplasmic protein T) and Yral (putative periplasmic pilin chaperone); or any subgroup
thereof. In one embodiment, X is a co-translational signal peptide or a post-translational
signal peptide. In one embodiment X is the signal sequence from FlgI (flgI sp). In another
particular ment, X is the signal sequence from pelB (pelB sp). In another embodiment,
X is a post-translational signal peptide. In another embodiment, X is selected from the group
consisting of the signal sequence from FlgI, NadA and pelB.
In one embodiment, the fusion proteins of formula (I) are defined wherein m is 1. In
another embodiment, m is 0.
In one particular embodiment, R1 and n are defined wherein (R1)n is 1 to 6 amino acids
enriched in small, usually hydrophilic, amino acids. Hydrophilic amino acids include glutamic
acid (E), aspartic acid (D) and asparagine (N).
In one ment, the fusion proteins of formula (I) are defined wherein n is selected
from the group consisting of 0, 1, 2 and 6. In one particular embodiment, R1 and n are defined
wherein (R1)n is selected from the group consisting of D, E, ATNDDD (SEQ ID NO. 178) and
MD, or any subset thereof.
In one particular embodiment, n is ed from the group consisting of 1, 2 and 6. In
one particular embodiment, n is 0.
In one embodiment, the fusion ns of formula (I) are defined wherein A is Protein E
from H. influenzae. In another ment, the fusion proteins of formula (I) are defined
wherein A is Protein E as encoded by an amino acid sequence selected from the group
consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,
SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID
NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19,
SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID
NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30,
SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID
WO 39225
NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41,
SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID
NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52,
SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any
subset of SEQ ID NO. 5 through SEQ ID NO. 57. In another embodiment, the fusion proteins
of formula (I) are defined wherein A is Protein E, wherein Protein E is approximately 75% to
100% identical to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In another
embodiment, A is Protein E wherein Protein E is imately 90% to 100% identical to the
Protein E amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, A is
Protein E wherein Protein E is at least 95% identical to the Protein E amino acid sequence set
forth in SEQ ID NO: 4. In additional embodiment, A is Protein E wherein Protein E is at least
95% cal to Protein E as set for in any of SEQ ID NO. 4 — SEQ ID NO. 57. In a ular
embodiment, A is Protein E having the amino acid sequence set forth in SEQ ID NO. 4.
In another embodiment, the fusion proteins of formula (I) are d wherein A is an
immunogenic fragment of Protein E from H. influenzae. In another embodiment, A is an
immunogenic fragment of n E wherein Protein E has an amino acid sequence selected
from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,
SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.
13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ
ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24,
SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID
NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35,
SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID
NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46,
SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID
NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO.
57; or any subset of SEQ ID NO. 4 through SEQ ID NO. 57. In another embodiment, A is an
immunogenic fragment of Protein E, wherein Protein E is approximately 75% to 100%
identical to the amino acid sequence set forth in SEQ ID NO: 4. In r embodiment, A is
an immunogenic fragment of Protein E, n Protein E is approximately 90% to 100%
identical to SEQ ID NO. 4. In an additional embodiment, A is an immunogenic fragment of
Protein E, wherein Protein E is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO.
57. More specifically, in one embodiment, A is an immunogenic fragment of Protein E,
n Protein E is 93% to 100% identical to SEQ ID NO. 124. In a particular embodiment,
A is an immunogenic fragment of Protein E wherein Protein E is SEQ ID NO. 4.
In another embodiment, A is an immunogenic fragment of Protein E from H. influenzae
selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122),
amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4
(SEQ ID NO. 124), amino acids 20—160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids
22-160 of SEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, A is an immunogenic
fragment of Protein E from H. influenzae selected from the group consisting of amino acids
17-160 ofSEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 ofSEQ ID NO. 4 (SEQ ID
NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20—160 of
SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126),
amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID
NO. 4 (SEQ ID NO. 180). In a further embodiment, A is an immunogenic fragment of n
E from H. zae selected from the group consisting of amino acids 17-160 of SEQ ID NO.
4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids
-160 ofSEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 ofSEQ ID NO. 4 (SEQ ID
NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of
SEQ ID NO. 4 (SEQ ID NO. 180). More specifically, in one embodiment, A is SEQ ID NO.
124, amino acids 19-160 of SEQ ID NO. 4. In an additional embodiment, A is SEQ ID
NO.125, amino acids 20—160 of SEQ ID NO. 5. In another embodiment, A is immunogenic
fragment of Protein E from H. influenzae selected from the group consisting of amino acids
23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ
ID NO. 180).
Protein E - SEQ ID NO. 4
MK< T.TT.ST. ACSAQ QI A*ZQNDVKL APPTDVRSGY IQLVKNVNYY
DSfiS WVD\T QfiL’Q VHE'DA VVNLDKGLYV YPEP<RYARS VQQYKILNCA
NY-ILTQVQTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTT. YWAAQ CAN
VDK <
Amino acids 17-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 122
SAQ QI A*1QNDVKL APPTDVRSGY :RLVKNVNYY
DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA
NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH DTTL YNAAQ CAN
VDK<
Amino acids 18-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 123
AQ QIAfiQNDVKL APPTDVRSGY :QLVKNVNYY
DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA
VQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTL YNAAQ CAN
YGEAFSVDK<
Amino acids 19-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 124
Q QIAfiQNDVKL APPTDVRSGY :QLVKNVNYY
DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA
NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTL YNAAQ CAN
YGEAFSVDK<
Amino acids 20—160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 125
QIAfiQNDVKL APPTDVRSGY :RLVKNVNYY
DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS LNCA
NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTL YNAAQ CAN
YGEAFSVDK<
Amino acids 22-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 126
KAEQNDVKL APPTDVRSGY :RLVKNVNYY
DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA
NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH DTTL YNAAQ CAN
YGEAFSVDK<
Amino acids 23-160 of n E from SEQ ID NO. 4 - SEQ ID NO. 179
AEQNDVKL APPTDVRSGY :RLVKNVNYY
DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA
2012/050236
NY-ILTQVQTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTT. YNAAQ CAN
YGEAFSVDK <
Amino acids 24-160 Protein E from SEQ ID NO. 4 - SEQ ID NO. 180
EQNDVKL APPTDVRSGY IRLVKNVNYY
DSfiS WVDW QfiL’Q VHE'DA VVNLDKGLYV YPEP<RYARS LNCA
NY-ILTQVQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTT. YWAAQ CAN
YGEAFSVDK <
In another embodiment, the fusion proteins of formula (I) are defined wherein A is PilA
from H. influenzae. In another embodiment, the fusion ns of formula (I) are defined
wherein A is PilA from H. influenzae having an amino acid ce selected from the group
consisting ofSEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO.
62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ
ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73,
SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID
NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84,
SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID
NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95,
SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID
NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO.
106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO.111,
SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116,
SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121;
or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In another embodiment, A is PilA
wherein PilA is approximately 80% to 100% cal to SEQ ID NO. 58. In another
ment, A is PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ
ID NO. 121. In a particular embodiment, A is PilA of SEQ ID NO. 58.
In another embodiment, the fusion proteins of formula (I) are defined wherein A an
immunogenic fragment of PilA from H. influenzae. In another embodiment, A is an
immunogenic fragment of PilA wherein PilA is approximately 80% to 100% identical to SEQ ID
NO. 58. For example, A is an immunogenic fragment of PilA wherein PilA has an amino acid
ce selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO.
60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ
ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71,
SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID
NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82,
SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID
NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93,
SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID
NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO.
104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109,
SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114,
SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119,
SEQ ID NO. 120 and SEQ ID NO. 121; or any subset SEQ ID NO. 58 through SEQ ID NO.
121. In an additional embodiment, A is an immunogenic nt of PilA wherein PilA is at
least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment,
A is an immunogenic fragment of PilA from H. influenzae strain 86-028NP n PilA is
SEQ ID NO. 58.
PilA from H. influenzae strain 86-028NP - SEQ ID NO. 58
MKLTTQQTL< KGld'TT. 41T.M V A A T.AT A BSYQNYT KKAAVSELLQ
ASAPYKADVE LCVYSTWETT NCTGGKNG A AD TTAKGYV KSVTTSWGA:
TVKGDGTT.A\T M*'.Y T.QATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ
In another embodiment, A is an genic fragment of PilA approximately 75% to
100% identical to SEQ ID NO. 127. More specifically, in one embodiment A is SEQ ID NO.
127, a fragment consisting of amino acids 40-149 of SEQ ID NO. 58.
Amino acids 40-149 of PilA from H. influenzae strain NP - SEQ ID NO. 127.
T KKAAVSELLQ
ASAPYKADVE LCVYSTNETT NCTGGKNG A A3 V KSVTTSWGA:
TVKGDGTT.A\T M*'.Y T.QATGN AATGVTWTTT C(GTDASLFP ANFCGSVTQ
In another embodiment, A is an immunogenic fragment of PilA consisting of amino acids
40-149 from any of SEQ ID NO. 58 — SEQ ID NO. 121. In an additional embodiment, A is an
genic fragment at least 95% identical to amino acids 40-149 from any of SEQ ID NO.
58—SEQ ID NO.121.
In one ment, the fusion proteins of formula (I) are d wherein Y is selected
from the group consisting of GG, SG and SS. In another embodiment, the fusion proteins of
formula (I) are defined wherein Y is GG or SG. In one particular embodiment, Y is GG.
In one embodiment, the fusion proteins of formula (I) are defined wherein o is 1. In
another embodiment, o is 0.
In one embodiment, the fusion proteins of formula (I) are defined wherein B is PilA from
H. influenzae or an immunogenic fragment of PilA from H. influenzae when A is n E from
H. zae or an immunogenic fragment of Protein E from H. influenzae. For example, B is
PilA from H. influenzae strain 86-028NP. In another ment, B is PilA from H. influenzae
having an amino acid sequence selected from the group consisting of SEQ ID NO. 58, SEQ
ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64,
SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID
NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75,
SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID
NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86,
SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID
NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97,
SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ
ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID
NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO.
113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118,
SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQ ID NO. 58
through SEQ ID NO. 121. In another embodiment, B is PilA wherein PilA is approximately
80% to 100% identical to SEQ ID NO. 58. In another embodiment, B is PilA wherein PilA is at
least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment,
B is PilA ofSEQ ID NO. 58.
In another embodiment, B is PilA wherein PilA is at least 95% identical to any of SEQ ID
NO. 58 — SEQ ID NO. 121 and A is PE wherein PE is at least 95% identical to any of SEQ ID
NO. 4 — SEQ ID NO. 57.
In another embodiment, the fusion proteins of formula (I) are defined n B is an
immunogenic fragment of PilA from H. influenzae when A is an immunogenic fragment of
Protein E from H. influenzae. For example, B is an immunogenic fragment of the PilA from H.
influenzae strain 86-028N P. In another embodiment, B is an genic fragment of PilA
wherein PilA is imately 80% to 100% identical to SEQ ID NO: 58. In another
embodiment, B is an immunogenic fragment of PilA wherein PilA has an amino acid selected
from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO.
61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ
ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72,
SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID
NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83,
SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID
NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94,
SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID
NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO.
105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110,
SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115,
SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and
SEQ ID NO. 121; or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In another
embodiment, B is an immunogenic nt of PilA wherein PilA is at least 95% identical to
any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment, B is an immunogenic
fragment of PilA from H. influenzae wherein PilA has the amino acid sequence set forth in
SEQ ID NO. 58. In another embodiment, B is an immunogenic fragment of PilA consisting of
amino acids 40-149 from any of SEQ ID NO. 58 — SEQ ID NO. 121. More specifically, in one
embodiment B is the fragment of PilA as set forth in SEQ ID NO. 127. In an additional
embodiment, B is an immunogenic fragment at least 95% identical to amino acids 40-149 of
any of SEQ ID NO. 58 — SEQ ID NO.121.
In one particular embodiment, B is the fragment of PilA as set forth in SEQ ID NO. 127
and A is an immunogenic fragment of n E selected from the group ting of SEQ ID
NO. 122, SEQ ID NO. 124, SEQ ID NO. 125 and SEQ ID NO. 126. More particularly, B is the
fragment of PilA as set forth in SEQ ID NO. 127 and A is the fragment of Protein E as set forth
in SEQ ID NO. 124, amino acids 19-160 of n E from SEQ ID NO. 4. In another
embodiment, B is the fragment of PilA as set forth in SEQ ID NO. 127 and A is the fragment of
Protein E as set forth in SEQ ID NO. 125.
In another embodiment, B is an immunogenic fragment of PilA wherein PilA is at least
95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121 and A is an immunogenic fragment
of PE wherein PE is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO. 57.
In another embodiment, the fusion proteins of formula (I) are defined wherein B is
Protein E from H. influenzae when A is PilA from H. zae. For example, B is Protein E
having an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID
NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID
NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16,
SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID
NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,
SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID
NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38,
SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID
NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49,
SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID
NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 h SEQ ID
NO. 57. In another embodiment, the fusion proteins of formula (I) are defined wherein B is
Protein E wherein Protein E is imately 75% to 100% cal to the Protein E amino
acid sequence set forth in SEQ ID NO: 4. In another embodiment, B is Protein E wherein
Protein E is imately 90% to 100% cal to the Protein E amino acid sequence set
forth in SEQ ID NO: 4. For example, B is Protein E wherein Protein E is at least 95% identical
to Protein E as set forth in SEQ ID NO. 4. In another embodiment, B is Protein E wherein
Protein E is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO. 57. In a particular
embodiment, B is Protein E having the amino acid sequence set forth in SEQ ID NO. 4.
In another embodiment, the fusion proteins of formula (I) are d wherein B is an
immunogenic fragment of Protein E from H. zae when A is an immunogenic fragment of
PilA from H. influenzae. For example, B is an immunogenic fragment of Protein E wherein
Protein E has an amino acid sequence selected from the group consisting of SEQ ID NO. 4,
SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10,
SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID
NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21,
SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID
NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32,
SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID
NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43,
SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID
NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54,
SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 through
SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are d n
B is an immunogenic fragment of Protein E wherein Protein E is approximately 75% to 100%
identical to the Protein E amino acid sequence set forth in SEQ ID NO. 4. In another
embodiment, B is an immunogenic fragment of Protein E wherein n E is approximately
90% to 100% identical to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In a
particular embodiment, B is an immunogenic fragment of Protein E having the amino acid
sequence set forth in SEQ ID NO. 4. In an additional embodiment, B is an immunogenic
fragment of Protein E, wherein Protein E is at least 95% cal to any of SEQ ID NO. 4 —
SEQ ID NO. 57.
In another embodiment, B is a nt of Protein E from H. influenzae selected from
the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids
18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID
NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 of
SEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, B is an immunogenic fragment of
Protein E from H. zae selected from the group consisting of amino acids 17-160 of SEQ
ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino
acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ
ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of
SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO.
180). More specifically, in one embodiment, B is the fragment of Protein E as set forth in SEQ
ID NO. 123, amino acids 18-160 of SEQ ID NO. 4.
In one particular embodiment B is an immunogenic fragment of Protein E as set forth in
SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4 when A is an immunogenic nt
of PilA as set forth in SEQ ID NO. 127.
In one embodiment, the fusion proteins of a (I) are defined wherein p is 0. In
another embodiment, the fusion proteins of formula (I) are defined wherein p is 1.
WO 39225
In one embodiment, the fusion protein of formula (I) is selected from the group consisting
of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144,
SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184,
SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194,
SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 and SEQ ID NO. 204;
or any subset thereof. In another embodiment, the fusion protein of formula (I) is
approximately 95% cal to any of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140,
SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150,
SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190,
SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200,
SEQ ID NO. 202 or SEQ ID NO. 204.
Fusion proteins of formula (I) are useful as immunogens in subjects such as mammals,
particularly humans. In particular, the fusion proteins of formula (I) are useful in inducing an
immune se against H. influenzae in subjects, particularly humans. More specifically,
the fusion proteins of formula (I) are useful in the treatment or prevention of otitis media and/or
AECOPD and/or pneumonia.
The present invention relates to immunogenic compositions sing Protein E from
H. influenzae (or an immunogenic nt thereof) and PilA from H. influenzae (or an
immunogenic fragment thereof), and immunogenic compositions sing fusion proteins of
Protein E from H. influenzae (or an immunogenic fragment thereof) and PilA from H.
influenzae (or an immunogenic fragment thereof). The present invention also relates to
vaccines comprising such genic compositions and therapeutic uses of the same.
In one embodiment, the immunogenic compositions comprise Protein E from H.
influenzae (or an immunogenic fragment thereof) and PilA from H. influenzae (or an
immunogenic fragment thereof). Protein E may be SEQ ID NO. 4 or a Protein E sequence at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 4.
The immunogenic nt of Protein E may be SEQ ID NO. 122, SEQ ID NO. 123,
SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126, or a sequence having at least 90%,
95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NO. 122, SEQ ID NO.
123, SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126. The immunogenic nt of
Protein E may be SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125,
SEQ ID NO. 126, SEQ ID NO. 179 or SEQ ID NO. 180 or a sequence having at least 90%,
95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NO. 122, SEQ ID NO.
123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQ ID NO. 179 or SEQ ID NO.
180. Amino acid differences have been described in Protein E from various Haemophi/us
species when compared to Protein E from Haemophi/us influenzae Rd as a reference .
Microbes & Infection gendum to “Identification of a novel Haemophi/us influenzae protein
important for adhesion to epitheIia cells” [Microbes Infect. 10 (2008) 87-97], available online
July 6, 2010, “Article in Press”) provides a sequence for Protein E from H. influenzae strain
772. WO2002/28889 provides a sequence for Protein E from H. zae strain 12085.
Protein E contains an epithelial cell binding region (PKRYARSVRQ YKILNCANYH
LTQVR, SEQ ID NO. 128) that has been reported to be conserved among more than 100
clinical NTHi isolates, encapsulated H. influenzae, and e collection strains analyzed
(Singh et al, J. Infect. Dis. 201(3):414-9 (2010)). Singh et al. reported that Protein E was
highly ved in both NTHi and encapsulated H. zae (96.9% — 100% identity without
the signal e). In one embodiment, the fragment of Protein E comprises the binding
region of SEQ ID NO. 128 (PKRYARSVRQ YKILNCANYH LTQVR).
PilA is a conserved adhesin expressed in vivo. Full length comparison of 64 sequences
of PilA from Haemophi/us influenzae demonstrated approximately 80% to 100% identity.
In another embodiment, the immunogenic composition comprises a fusion protein as
defined by formula (I).
In one embodiment, the present immunogenic compositions may be stered with
other antigens from H. influenzae. For e, the PE and PilA or the fusion protein of
a (I) may be administered with n D from H. zae. Protein D may be as
described in WO91/18926. In another embodiment, the immunogenic composition may
include the fusion protein of formula (I) and Protein D from H. influenzae.
In another embodiment, the immunogenic compositions of the invention may be
administered with additional antigens from other bacterial species also known to cause otitis
media, AECOPD or pneumonia.
The amount of the immunogenic composition which is required to achieve the desired
therapeutic or biological effect will depend on a number of factors such as the use for which it
is ed, the means of administration, the recipient and the type and severity of the
condition being d, and will be ultimately at the discretion of the attendant physician or
veterinarian. In general, a typical dose for the treatment of a condition caused in whole or in
part by H. influenzae in a human, for instance, may be expected to lie in the range of from
about 0.003 mg to about 0.090 mg. More specifically, a typical dose for the treatment of a
ion caused wholly or in part by H. influenzae in a human may lie in the range of from
about 0.01 mg to about 0.03 mg of fusion n. The immunogenic composition may contain
additional antigens; a typical dose for the treatment of a condition caused wholly or in part by
H. influenzae in a human may lie in the range of from about 0.01 mg to about 0.03 mg for
each additional antigen. This dose may be administered as a single unit dose. Several
separate unit doses may also be administered. For example, separate unit doses may be
administered as separate priming doses within the first year of life or as separate booster
doses given at regular intervals (for example, every 1, 5 or 10 .
Formulations comprising the immunogenic compositions of the invention may be
d for administration by an appropriate route, for example, by the intramuscular,
sublingual, transcutaneous, intradermal or intranasal route. Such formulations may be
ed by any method known in the art.
The immunogenic compositions of the t invention may additionally comprise an
adjuvant. When the term “adjuvant” is used in this ication, it refers to a substance that is
administered in conjunction with the genic composition to boost the patient’s immune
se to the immunogenic component of the composition.
Suitable adjuvants e an aluminum salt such as aluminum hydroxide gel or
aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc, or
may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or
anionically derivatized saccharides, or polyphosphazenes. In one embodiment, the fusion
protein, PE or PilA may be adsorbed onto ium phosphate. In another embodiment, the
fusion protein, PE or PilA may be adsorbed onto aluminium hydroxide. In a third embodiment,
alum may be used as an adjuvant.
Suitable adjuvant systems which promote a predominantly Th1 response include: non-
toxic derivatives of lipid A, Monophosphoryl lipid A (MPL) or a derivative thereof, particularly 3-
de-O-acylated monophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A); and
a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A,
er with either an aluminum salt (for instance aluminum phosphate or aluminum
hydroxide) or an oil-in-water on. In such combinations, antigen and 3D-MPL are
contained in the same particulate structures, allowing for more efficient delivery of antigenic
and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance
the immunogenicity of an alum-adsorbed antigen (Thoelen et al. Vaccine (1998) -14;
EP 689454-81).
SO1 is an Adjuvant System containing MPL (3-O-desacyl-4’- monophosphoryl lipid A),
QS21 (Qui/laja saponaria Molina, fraction 21) Antigenics, New York, NY, USA) and liposomes.
A8018 is an Adjuvant System ning MPL, QS21 and liposomes (50 ug MPL and 50 ug
QS21). ASO1E is an Adjuvant System containing MPL, QS21 and mes (25 ug MPL and
ug QS21). In one embodiment, the immunogenic composition or vaccine ses ASO1.
In another embodiment, the immunogenic composition or vaccine comprises ASO1B or
ASO1 E. In a particular embodiment, the immunogenic composition or vaccine comprises
ASO1E.
A803 is an Adjuvant System containing d-Tocopherol and squalene in an oil/water (o/w)
emulsion. ASO3A is an Adjuvant System containing d-Tocopherol and squalene in an o/w
emulsion (11.86 mg tocopherol). A8033 is an Adjuvant System containing pherol and
squalene in an o/w emulsion (5.93 mg tocopherol). A8030 is an Adjuvant System containing d-
Tocopherol and squalene in an o/w emulsion (2.97 mg tocopherol). In one embodiment, the
immunogenic composition or vaccine comprises A803.
A804 is an Adjuvant System containing MPL (50 ug MPL) adsorbed on an aluminum
salt (500 ug Al3+ ). In one embodiment, the immunogenic composition or vaccine comprises
ASO4.
A system involving the use of QS21 and 3D-MPL is disclosed in WO 94/00153. A
composition wherein the QS21 is quenched with cholesterol is disclosed in WO 39. An
onal adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water
emulsion is described in WO 95/17210. In one embodiment the genic ition
additionally comprises a saponin, which may be QS21. The formulation may also comprise an
oil in water emulsion and tocopherol (WO 95/17210). Unmethylated CpG containing
oligonucleotides (WO 55) and other immunomodulatory oligonucleotides (WO 0226757
and WO 03507822) are also preferential inducers of a TH1 response and are suitable for use
in the present invention.
Additional adjuvants are those selected from the group of metal salts, oil in water
emulsions, Toll like receptor agonists, (in ular Toll like receptor 2 agonist, Toll like
or 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8
agonist and Toll like receptor 9 agonist), saponins or combinations thereof.
The present invention provides a s for preparing an immunogenic
composition comprising combining a fusion protein of formula (I) with an adjuvant.
The present invention r provides a vaccine ning an immunogenic
composition of the invention and a pharmaceutically acceptable excipient.
Possible excipients e arginine, pluronic acid and/or polysorbate. In a preferred
embodiment, polysorbate 80 (for example, TWEEN® 80) is used. In a further embodiment, a
final concentration of about 0.03% to about 0.06% is used. ically, a final concentration
of about 0.03%, 0.04%, 0.05% or 0.06% polysorbate 80 (w/v) may be used.
The present invention provides a process for preparing an genic composition or
vaccine comprising combining a fusion protein of formula (I) with a ceutically
acceptable excipient.
The present invention also provides nucleic acids encoding the proteins of the invention.
The term “nucleic acid” refers to a polymeric form of nucleotides. tides can be
ribonucleotides, deoxyribonucleotides, or modified forms of either ribonucleotides or
deoxyribonucleotides. The term includes single and double forms of DNA. The nucleic acids
are preferably substantially free from other nucleic acids.
The present invention provides a process of producing nucleic acids of the invention.
Nucleic acids of the invention may be ed by methods known by those skilled in the art.
For example, the c acids of the invention may be synthesized in part or in whole. The
nucleic acids may be ed by digesting longer amino acids orjoining shorter amino acids.
The following examples are intended for illustration only and are not intended to limit the
scope of the invention in any way.
In the examples, the ing terms have the designated meaning:
6xhis = six histidines;
xg = centrifugal force r gravities)
ATP = adenosine triphosphate;
BCA = bicinchoninic acid;
BSA = bovine serum albumin;
°C = degrees Celsius;
CaClz = calcium chloride;
CV = column volume;
DNA = deoxyribonucleic acid;
DSC = ential scanning calorimetry;
DTT = dithiothreitol;
dNTP = deoxynucleoside triphosphate;
EDTA = ethylenediaminetetraacetic acid;
FT = flow through;
HCI = hydrogen chloride;
His = his = histidine;
HEPES = 4-(2-hydroxyethyl)piperazineethanesulfonic acid;
IMAC = lized metal ty chromatography;
IPTG = isopropyl [3-Dthiogalactopyranoside;
KCI = potassium chloride;
KZHPO4 = dibasic potassium phosphate;
KHZPO4 = monobasic potassium phosphate;
LDS = lithium dodecyl sulfate;
L = liter;
MES = 2—(N-morpholino)ethanesulfonic acid;
MgClz = magnesium chloride;
ml = iter;
RPM = revolutions per minute;
min = minute;
mM = millimolar;
uL = microliter;
NaCl = sodium chloride;
NazHPO4 = dibasic sodium phosphate;
NaHzPO4 = monobasic sodium phosphate;
ng = nanogram;
nm = nanometer;
O/N = overnight;
PBS = phosphate ed saline;
PCR = polymerase chain reaction;
SB = sample buffer;
sec = second;
w/v = weight/volume.
EXAMPLES
Example 1: Fusion proteins
Fusion proteins were produced with different signal es and amino acid linker
sequences. These fusion proteins allowed for secretion of both Protein E and PilA (or
fragments thereof) without being restricted to a single bacterial . The fusion protein is
released into the periplasm after removal of the heterologous signal peptide by a signal
peptide peptidase. Fusion protein purified from the bacteria does not n the
heterologous signal peptide. “Purified” proteins are removed from the bacteria and lack the
signal peptide.
The following table describes fusion protein constructs made.
WO 39225
Table 3: Fusion Protein Constructs containing PiIA and n E.
N-terminal -------------------------------------------------------------------------------C-Terminal
LVL312 E PilA fragment ProtE fragment GGHHHHHH
(A.A.: 40—149 of SEQ ID NO. (AA: 18 to 160 of SEQ ID NO. 4, SEQ
58, SEQ ID NO. 127) ID NO.123)
AA. 1 19 21 130 133 275 276 283
LVL291 pelB sp ProtE fragment G PilA fragment GGHHHHHH
(AA: 19 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO.
NO. 124 58, SEQ ID NO. 127
A A 1 22 23 164 167 276 277 284
LVL268 pelB sp ProtE fragment G PilA fragment GGHHHHHH
(AA: 20 to 160 of SEQ ID NO. 4, SEQ G (A.A.: 40—149 of SEQ ID NO.
ID NO. 125 58, SEQ ID NO. 127
AA. 1 22 24 164 167 276 277 284
LVL269 nadA sp AT ProtE fragment G PilA fragment GGHHHHH
ND (AA: 22 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. H
DD NO. 12- 58, SEQ ID NO. 127
AA. 1 23 24-29 30 168 171 280 281 288
LVL270 MH ProtE fragment G PilA fragment
HH (AA: 17 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 58,
HH NO. 122) SEQ ID NO. 127)
AA. 1 7 8 151 154 263
LVL315 pelB ProtE fragment GGHHHHHH
sp (AA: 22 to 160 of SEQ ID NO. 4, SEQ
ID NO. 126)
1 22 25 163 166 275 276 283
LVL317 pelB sp ProtE fragment G PilA fragment
(AA: 19 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID
NO. 124) NO. 58, SEQ ID NO. 127)
AA. 1 22 23 164 167 27
LVL318 pelB sp | ProtE fragment G PilA fragment
(AA: 22 to 160 of SEQ ID NO. 4, SEQ G (A.A.: 40—149 of SEQ ID
ID NO. 126) NO. 58, SEQ ID NO.
AA. 1 22 25 163 166 275
LVL702 pelB sp ProtE fragment GGHHHHHH
NO. 125) NO. 58, SEQ ID NO.
127)
AA. 1 22 23 163 166 275 283
LVL736 pelB sp ProtE fragment G PilA fragment GG
(AA: 17 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 58, HH
NO. 122) SEQ ID NO. 127) HH
AA. 1 22 23 166 169 278 286
LVL737 pelB Sp ProtE fragment G PilA ragment GGH
(AA: 18 to 160 of SEQ ID NO. 4, SEQ ID (A.A.: 40—149 of SEQ ID NO. 58, HHH
--I-
22 23 277 285
LVL738 pelB sp ProtE fragment G PilA fragment GGHHHHHH
(A.A.: 22 to 160 of SEQ ID NO. 4, G (A.A.: 40—149 of SEQ ID
SEQ ID NO. 126) NO. 58, SEQ ID NO.
127)
2223164
LVL739 pelB sp ProtE nt G PilA nt GGHHHHHH
(A.A.: 23 to 160 of SEQ ID NO. 4, (A.A.: 40—149 of SEQ
SEQ ID NO. 179) ID NO. 58, SEQ ID
NO. 127)
22 23 160163 272
LVL740 pelB sp ProtE fragment G PilA fragment GGHHHHHH
(A.A.: 24 to 160 of SEQ ID NO. (A.A. 40—149 of SEQ
4, SEQ ID NO. 180) ID NO. 58, SEQ ID
NO. 127)
22 23 159162 27
LVL735 pelB sp ProtE fragment G PilA fragment
(A.A.: 20 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID
NO. 125) NO. 58, SEQ ID NO. 127)
22 23 163
LVL778 pelB sp ProtE fragment G PilA fragment
(A.A.: 17 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID
NO. 122) NO. 58, SEQ ID NO. 127)
22 23
LVL779 pelB sp ProtE fragment G PilA fragment
(A.A.: 18 to 160 of SEQ ID NO. 4, SEQ ID G (A.A. 40—149 of SEQ ID
) NO. 58, SEQ ID NO.127)
22 23 168 277
LVL780 pelB Sp ProtE fragment G PilA fragment
(A.A.: 22 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID
NO. 126) NO. 58, SEQ ID NO. 127)
22 23 161 164 273
LVL781 pelB sp ProtE fragment G PilA fragment
(A.A.: 23 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID
NO.179) NO. 58, SEQ ID NO. 127)
22 23 160 163 272
LVL782 pelB sp ProtE fragment G PilA fragment
(A.A.: 24 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID
NO. 180) NO. 58, SEQ ID NO. 127)
22 23 159 1
sp = signal peptide; AA. = amino acid
The DNA and amino acid sequences for each of the signal peptides and plasmids listed in
Table 3 are set forth below.
SIGNAL SEQUENCES:
pe/B signal peptide (DNA) — SEQ ID NO. 129:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcc
pe/B signal peptide (Amino Acid) - SEQ ID NO. 130:
MKYLLPTAAA GLLLLAAQPA MA
FIgI signal peptide (DNA) - SEQ ID NO. 131:
atgattaaatttctctctgcattaattcttctactggtcacgacggcggctcaggct
FIgI signal peptide (Amino Acid) - SEQ ID NO. 132:
M Kb'TSAT. T. T.T.VTTAAQA
NadA signal peptide (DNA) - SEQ ID NO. 133:
atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcgcactggca
NadA signal peptide (Amino Acid) - SEQ ID NO. 134:
MKHFPSKVLT TA: LAT FCSG ALA
FUSION PROTEIN CONSTRUCT SEQUENCES:
The single underlined portion of the amino acid sequences is from PiIA from Haemophi/us
influenzae strain NP. The embolded underlined portion of the amino acid sequences
was derived from Protein E from Haemophi/us influenza strain 772.
LVL312 (DNA) - SEQ ID NO. 135:
aaatttctctctgcattaattcttctactggtcacgacggcggctcaggctgagactaaaaaagcagcggtatctgaattactg
caagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatg
cagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggat
ggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaac
ctctttatttccagcaaatttttgcggaagtgtcacacaaggcggcgcgcagattcagaaggctgaacaaaatgatgtgaa
gctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtg
gataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgca
cgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt
ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg
cgaactatggtgaagcattttcagttgataaaaaaggcggccaccaccaccaccaccactaa
LVL312 (protein): (flgI sp)(E)(PiIA aa 40-149)(GG)(ProtE aa 18-160)(GGHHHHHH) - SEQ ID
NO. 136
M KETSAL L LLVTTAAQA? TKIAAVS?LL QASAPYKADV ELCVYSTNET
KNG RAD TTAKGY VKSVTTSWGA :TVKGDGTLA.WM£Y LQATG
NAATGVTWTT TC<GTDASLF PANFCGSVTQ GGAQ QIAfiQ WDVKLAPPTD
VRSGYIQLVK NVWYY DSfiS WVDW fiB VNLD <GLYVYPEP<
RYARSV? YK :LWCANYiLT VQTDFYDEF WG GLRAAP< < KKHTLSLT
PDTTLYWAA AF SVDK<GG€€H HHH
LVL291 (DNA) - SEQ ID NO. 137:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaaca
aaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtga
atcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcc
taaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattt
tggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgc
tcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa
gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtatt
gcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggc
acattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacgga
tgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaa
LVL291 (Protein)(pe|B sp)(ProtE aa 19-160)(GG)(PiIA aa40—149)(GGHHHHHH) - SEQ ID NO.
TAAA GLLLLAAQPA NA IAfi N DVKLAPPTDV RSGYIQLVKN
VNYY DSfiS WVDW fiB V NLDK GLYVYPEP<R YARSV? YK:
LNCANYiLT VQTDFYDEFW G GLRAAP<K KKHTLSLTP DTTLYWAA I
"CANYG?AFS VDK<GGT<KA AVSELLQASA PYKADVELCV YSTNETTNCT
GGKNG RAD VKSV ITVK GDGTLANMfiY AAT
GVTWTTTC<G TDASLFPANF CGSVTQGGHH HiHH
LVL268 (DNA) - SEQ ID NO. 139:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgatattcagaaggctgaaca
aaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtga
atcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcc
taaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattt
tggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgc
tcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa
gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtatt
gcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggc
acattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacgga
tgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL268 (protein): (peIB sp)(D)(ProtE aa 20—160)(GG)(PiIA aa40-149)(GGHHHHHH) - SEQ ID
NO. 140:
MKYLLPTAAA GLLLLAAQPA NAD IAfi N DVKLAPPTDV RSGYIQLVKN
VNYY DSfiS WVDW fiB V PEDAVVNLDK GLYVYPEP<R YARSV? YK:
LNCANYiLT VQTDFYDEFW G GLRAAP<K KKHTLSLTP AA I
"CANYG?AFS VDK<GGT<KA AVSELLQASA PYKADVELCV YSTNETTNCT
GGKNG RAD TTA<GYV<SV TTSWGAITVK WMfiY LQATGNAAT
GVTWTTTC<G TDASLFPANF CGSVTQGGHH HiHH
LVL269 (DNA) - SEQ ID NO. 141:
atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcgcactggcagccacaaacgacgacg
ataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattatt
acatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtat
gtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgat
ttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaac
gctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtat
ctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtg
gaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagta
gatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttg
caaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaa
LVL269 (protein): (nadA sp)(ATNDDD)(ProtE aa 22-160)(GG)(PiIA aa 40-149)(GGHHHHHH)
- SEQ ID NO.142
MKHFPSKVLT TAILATFCSG DDDK A:L*J ZDVKLAP PTDVRSGYIQ
LVKNVNYY D SfiS WVDW fl B VHEDAVV NLDKGLYVYP EP<RYARSVQ
YKILNCANY {LT VRTDFY DEFWG GLRA AP<K KKHTL SLTPDTTLYW
AA CANYG ?AFSV3<<GG T<KAAVSELL KADV ELCVYSTNET
TNCTGGKNG AAD TTA<GY V<SVTTSWGA :TVKGDGTLA.WM£Y LQATG
NAATGVTWTT TC<GTDASLF PANFCGSVTQ GGHHHiHH
LVL270 (DNA) - SEQ ID NO. 143:
atgcaccaccaccaccaccacagcgcgcagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacg
aagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgta
cattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttg
aattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagca
aaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagtt
gataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgt
atatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaa
aatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacag
gtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcac
acaataa
LVL270 (protein): (MHHHHHH)(ProtE aa 17-160)(GG)(PiIA aa40-149) - SEQ ID NO. 144.-
WHiiiHHSA IAfi NDVK LAPPTDVQSG YIQLVKNVNY Y DSfiS WVD
W fiB VHED AVVNLDKGLY VYPEP<RYAR SV? YKILNC ANYiLT VAT
DFYDEFWG G LRAAP<K KK HTLSLTPDTT LYWAA CA NYGEAFSVDK
jGGT<KAAVS ELLQASAPYK VYST TGGK NG AAD TTA
<GYV<SVTTS WGAITVKGDG TLAWMfiY LQ ATGNAATGVT GTDA
SLFPANFCGS VTQ
LVL315 (DNA) - SEQ ID NO. 145:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaa
tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg
atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa
cgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg
gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag
attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt
cagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca
gcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacat
tggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcc
tctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaa
LVL315 (protein): (peIB sp)(M D)(ProtE aa 22-160)(GG)(PiIA aa40—149)(GGHHHHHH) - SEQ
ID NO. 146:
MKYLLPTAAA GLLLLAAQPA MAMDKAE ND VKLAPPTDV? SGYIQLVKNV
NYY DSfiS W VD\T *ZL’ VH b'DAVVNLDKG P<RY ARSV? YKIL
NCANY-ILT V QTDFYDEFWG GLQAAP<K KKHTLSLTPD TTT.Y\TAA
AFSV DK<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG
GKNG AAD T TA<GYV<SVT TVKG DGTT.A\IM*ZY T.QATGNAATG
VTWTTTC<GT DASLFPANFC GSVTQGGHHH -IHH
LVL317 (DNA) - SEQ ID NO. 147:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaaca
tgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtga
atcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcc
taaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattt
tggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgc
tcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa
gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtatt
gcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggc
acattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacgga
tgcctctttatttccagcaaatttttgcggaagtgtcacacaataa
LVL317 (protein): (peIB sp)(ProtE aa 19-160)(GG)(PiIA aa40-149) - SEQ ID NO. 148:
MKYLLPTAAA GLLLLAAQPA NA IAfi N DVKLAPPTDV RSGYIQLVKN
VNYY DSfiS WVDW fiB V PEDAVVNLDK GLYVYPEP<R YARSV? YK:
iLT VQTDFYDEFW G GLRAAP<K KKHTLSLTP DTTLYWAA I
"CANYG?AFS VDK<GGT<KA AVSELLQASA ELCV YSTNETTNCT
GGKNG RAD TTA<GYV<SV TTSWGAITVK GDGTLAWMfiY LQATGNAAT
GVTWTTTC<G TDASLFPANF CGSVTQ
LVL318 (DNA) - SEQ ID NO. 149:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaa
tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg
atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa
cgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg
gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag
attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt
cttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca
gcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacat
tggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcc
tctttatttccagcaaatttttgcggaagtgtcacacaataa
LVL318 in): (peIB sp)(M D)(ProtE aa 22-160)(GG)(PiIA aa40-149) - SEQ ID NO. 150.-
MKYLLPTAAA GLLLLAAQPA MAMDKAL‘LJ ND VKLAPPTDVR VKNV
NYY DSfiS W VDW fiB VH EDAVVNLDKG LYVYPEP<RY ARSV? YKIL
NCANYiLT V QTDFYDEFWG GLRAAP<K KKHTLSLTPD TTLYWAA
CANYGEAFSV DK<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG
GKNG AAD T TA<GYV<SVT TSWGAITVKG DGTLAWMfiY AATG
VTWTTTC<GT DASLFPANFC GSVTQ
LVL702 (DNA) - SEQ ID NO. 181:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaa
tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg
atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa
gcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg
gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag
attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt
cagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca
gcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacat
tggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcc
tctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL702 (protein): (peIB sp)(ProtE aa 20-160)(GG)(PiIA aa40-149)(GGHHHHHH) - SEQ ID
NO. 182:
MKYLLPTAAA GLLLLAAQL’A MA IAfi ND VKLAPPTDV? SGYIQLVKNV
NYY DSfiS W VD\T *ZL’ VH b'DAVVNLDKG LYVYPEP<RY ARSV? YKIL
NCANY-ILT V QTDFYDEFWG GLRAAP<K KKHTLSLTPD TAA
CANYGEAFSV DK<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG
GKNG AAD T TA<GYV<SVT TSWGAITVKG DGTT.A\IM*ZY T.QATGNAATG
VTWTTTC<GT DASLFPANFC GSVTQGGHHH -IHH
LVL736 (DNA) - SEQ ID NO. 183:
tacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggc
tgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcga
tagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcct
gagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgat
gaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataa
tgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaatta
gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaa
atggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggg
gatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaagg
aacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL736 (protein): (peIB sp)(ProtE aa 17-160)(GG)(PiIA aa40—149)(GGHHHHHH) - SEQ ID
NO. 184:
TAAA GLLLLAAQPA MASAQ QIAfi QNDVKLAPPT DVRSGYIQLV
KNVNYY DSfi S WVDW fiB VHEDAVVNL DKGLYVYPEP {RYARSVQ Y
KILNCANYiL T VQTDFY37L‘L FWG GLRAAP {K {KHTLSL TPDTTLYWAA
CANYG?A GGT< KAAVSELLQA SAPYKADVEL CVYSTNETTN
CTGGKNG AA 3 TTA<GYV< SVTTSWGAIT LAWM fiY LQATGNA
ATGVTWTTTC {GTDASLFPA NFCGSVTQGG HHHiHH
LVL737 (DNA) - SEQ ID NO. 185:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctga
tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatag
tgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctga
gcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatga
attttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgc
tgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactg
caagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatg
gtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggat
ggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaac
ggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL737 (protein): (pe/B sp)(ProtE aa 18-160)(GG)(Pi/A aa40—149)(GGHHHHHH) - SEQ ID
NO. 186:
MKYLLPTAAA GLLLLAAQPA MAAQ QIAfiQ NDVKLAPPTD QLVK
NVNYY DSfiS WVDW fiB VHEDAVVNLD KGLYVYPEP< RYARSV? YK
:LNCANYiLT VQTDFYDEF WG GLRAAP< K {KHTLSLT WAA
CANYG?AF SVDK<GGT<K AAVSELLQAS APYKADVELC VYSTNETTNC
TGGKNG AAD TTA<GYV<S AITV KGDGTLAWMfi Y LQATGNAA
TGVTWTTTC< GTDASLFPAN FCGSVTQGGH HHiHH
WO 39225
LVL738 (DNA) - SEQ ID NO. 187:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgt
gaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctg
ggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgtta
tgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggaca
gggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatt
tgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagc
gccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcag
atataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggca
aatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatt
tccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL738 (protein): (pe/B sp)(ProtE aa 22-160)(GG)(Pi/A aa40—149)(GGHHHHHH) - SEQ ID
NO. 188:
MKYLLPTAAA GLLLLAAQPA MAKAL*J ZDVK LAPPTDVRSG YIRLVKNVNY
Y DSfiS WVD W fiB VHED AVVNLDKGLY VYPEP<RYAR SV? YKILNC
ANYiLT VQT DFYDEFWG G LRAAP<K KK HTLSLTPDTT LYWAA CA
NYGEAFSVDK <GGT<KAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK
NG AAD TTA VTTS KGDG TLAWMfiY LQ ATGNAATGVT
GTDA SLFPANFCGS VTQGGHHHiH H
LVL739 (DNA) - SEQ ID NO. 189:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaa
gctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtg
gataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgca
cgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt
ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg
cgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgcct
tataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatat
aaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaat
atggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttcc
agcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL739 (protein): (pe/B sp)(ProtE aa 23-160)(GG)(Pi/A 49)(GGHHHHHH) - SEQ ID
NO. 190.-
M<YLLPTAAA GLLLLAAQPA MARL*J ZDVKL APPTDVRSGY :QLVKNVNYY
DSfiS WVDW fiB VHEDA VVNLDKGLYV YPEP<RYARS V? YKILNCA
NYiLT VQTD FYDEFWG GL RAAP<K {KH TLSLTPDTTL YWAA CAN
YGEAFSVDK< GGT<KAAVSE.L LLQASAPYKA DVELCVYSTN ETTNCTGGKN
G RAD TTA< GYV<SVTTSW GAITVKGDGT LAWMfiY LQA TGNAATGVTW
TDAS LFPANFCGSV TQGGHHHiHH
LVL740 (DNA) - SEQ ID NO. 191:
tacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagct
ggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggat
aaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgt
tctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttg
cgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcg
aactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttat
aaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataac
cacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatg
gaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccag
caaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL740 (protein): (pe/B sp)(ProtE aa 24-160)(GG)(Pi/A aa40—149)(GGHHHHHH) - SEQ ID
NO. 192.-
M<YLLPTAAA GLLLLAAQPA MAIL*J ZDVKLA PPTDVQSGY: QLVKNVNYY:
iSfiS WVDWD fiB VHEDAV VNLDKGLYVY PEP<RYARSV Q YKILNCAN
YiLT VQTDF YDEFWG GLR AAP<K KKHT LSLTPDTTLY WAA CANY
GEAFSVDK<G GT<KAAVSEL LQASAPYKAD STNE TTNCTGGKNG
RAD TTA<G YV<SVTTSWG AITVKGDGTL AWMfiY LQAT VTWT
TTC<GTDASL GSVT QGGHHHiHH
LVL735 (DNA) - SEQ ID NO. 193:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaa
tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg
atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa
cgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg
gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag
attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt
cagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca
ataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacatt
ggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcct
ctttatttccagcaaatttttgcggaagtgtcacacaa
LVL735 (protein): (pe/B otE aa 20-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 194.-
MKYLLPTAAA GLLLLAAQL’A MA 521 A*1§2ND VKLAPPTDVR VKNV
NYY DSfiS W VD\T *ZL’ VH b'DAVVNLDKG LYVYPEP<RY ARSV? YKIL
NCANY-ILT V QTDFYDEFWG GLRAAP<K KKHTLSLTPD TTT.Y\TAA
AFSV D<<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG
GKNG AAD T TA<GYV<SVT TSWGAITVKG DGTT.A\IM*ZY T.QATGNAATG
VTWTTTC {GT DASLFPANFC GSVTQ
LVL778 (DNA) - SEQ ID NO. 195:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggc
tgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcga
tagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcct
gagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgat
gaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataat
gctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattac
tgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaat
ggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaagggg
atggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaagga
acggatgcctctttatttccagcaaatttttgcggaagtgtcacacaa
VV()2012/139225
LVL778 (protein): (pe/B sp)(ProtE aa )(GG)(Pi/A aa40—149) - SEQ ID NO. 196:
MKYLLPTAAA GLLLLAAQPA NASA IA*'. NDVKLAPPT DVQSGYIQLV
KNVNYY DSfi S WVDW fiB VNL DKGLYVYR {RYARSVQ Y
KILNCANYiL T VQTDFY37L‘L FWG GLRAAP {K KKHTLSL TPDTTLYWAA
CANYG?A FSV3K<GGT< KAAVSELLQA SAPYKADVEL CVYSTNETTN
G AA 3 TTA<GYV< SVTTSWGAIT VKG:DGTLAWM fiY A
ATGVTWTTTC {GTDASLFPA NFCGSVTQ
LVL779 (DNA) - SEQ ID NO. 197:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctga
tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatag
tgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctga
gcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatga
attttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgc
tgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactg
caagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatg
gtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggat
ggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaac
ggatgcctctttatttccagcaaatttttgcggaagtgtcacacaa
LVL779 (protein): (pe/B sp)(ProtE aa 18-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 198:
MKYLLPTAAA GLLLLAAQPA MAA I Afi NDVKLAPPTD VQSGYIQLVK
NVNYY DSfiS WVDW fiB VHEDAVVNLD KGLYVYPEP 4 ? YK
:LNCANYiLT VQTDFYDEF WG GLRAAP < K KKHTLSLT PDTTLYWAA
CANYG?AF SVDK<GGT<K AAVSELLQAS APYKADVELC VYSTNETTNC
TGGKNG AAD TTA<GYV<S VTTSWGAI:TV KGDGTLAWMZ4 Y LQATGNAA
TGVTWTTTC< GTDASLFPAN FCGSVTQ
LVL780 (DNA) - SEQ ID NO. 199.‘
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgt
gaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctg
ggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgtta
tgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggaca
gggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatt
tgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagc
taaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcag
atataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggca
aatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatt
tccagcaaatttttgcggaagtgtcacacaa
LVL780 (protein): (pe/B sp)(ProtE aa 22-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 200.-
MKYLLPTAAA GLLLLAAQPA MAKAES 2NDVK LAPPTDVQSG YIQLVKNVNY
Y DSfiS WVD \T *1? VHE'D AVVNLDKGLY VYPEPKRYAR SV? YKILNC
ANY-1LT VQT DFYDEFWG G LRAAP<K KK HTLSLTPDTT TIYWAA CA
NYGEAFSVDK <GGT<KAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK
NG RAD TTA <GYV<SVTTS \TGAITVKGDG TT.A\]M*'.Y T.Q ATGNAATGVT
WTTTC<GTDA SLFPANFCGS VTQ
LVL781 (DNA) - SEQ ID NO. 201:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaa
gctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtg
gataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgca
cgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt
gcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg
cgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgcct
tataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatat
aaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaat
tatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttcc
agcaaatttttgcggaagtgtcacacaa
LVL781 (protein): (pe/B sp)(ProtE aa 23-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 202.-
M<YLLPTAAA AQPA MAAE NDVKL APPTDVRSGY :QLVKNVNYY
DSfiS WVDW fiB VHEDA VVNLDKGLYV YPEP<RYARS V? YKILNCA
NYiLT VQTD FYDEFWG GL RAAP<K KKH TLSLTPDTTL YWAA CAN
YGEAFSV3<< AVSE.L LLQASAPYKA DVELCVYSTN ETTNCTGGKN
G AAD TTA< TTSW GAITVKGDGT LAWMfiY LQA TGNAATGVTW
TTTC<GTDAS LFPANFCGSV TQ
LVL782 (DNA) - SEQ ID NO. 203:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagct
ggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggat
aaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgt
tctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttg
cgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcg
aactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttat
aaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataac
cacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatg
gaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccag
caaatttttgcggaagtgtcacacaa
LVL782 (protein): (pe/B sp)(ProtE aa 24-160)(GG)(Pi/A 49) - SEQ ID NO. 204.-
TAAA GLLLLAAQPA MAIL‘LJ NDVKLA PPTDVRSGY: QLVKNVNYY:
DSfiS WVDW fiB VHEDAV VNLDKGLYVY PEP<RYARSV Q YKILNCAN
YiLT VQTDF YDEFWG GLR AAP<K {KHT LSLTPDTTLY WAA CANY
GEAFSVDK<G GT<KAAVSEL LQASAPYKAD VELCVYSTNE TTNCTGGKNG
AAD TTA<G YV<SVTTSWG AITVKGDGTL AWMfiY LQAT GNAATGVTWT
TTC<GTDASL FPANFCGSVT Q
WO 39225
The full length sequence for PE and PilA from which the above sequences were obtained are
set forth in SEQ ID NO. 4 (PE) and SEQ ID NO. 58 (PilA), respectively.
Example 2: Vector Construction and Transformation
Primers for amplifying PE from H. influenzae strain 772 were designed based on the
sequence of H. influenzae strain Hi Rd. The 5’ primer sequence contains one nucleotide
difference compared to the NTHi 772 sequence, introducing an amino acid difference at
position 24 when compared with the currently reported NTHi 772 genome sequence. Amino
acid #24 in the fusion protein constructs is E (glutamic acid) instead of K (lysine) as found in
NTHi 772.
DNA Sequence for PE from H. influenzae strain Rd. - SEQ ID NO. 151
atgaaaaaaattattttaacattatcacttgggttacttaccgcttgttctgctcaaatccaaaaggctgaacaaaatgatgtgaagctg
gcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggata
agccacaaattgtacattttgatgctgtggtgaatttagataggggattgtatgtttatcctgagcctaaacgttatgcacgttc
tgttcgtcagtataagattttgaattgtgcaaattatcatttaactcaaatacgaactgatttctatgatgaattttggggacagggtttgcg
ggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcaaat
aaagcattttcagttgataaaaaataa
n Sequence for PE from H. influenzae strain Rd. - SEQ ID NO. 152
M<K T.TT.ST. GT.T.TACSAQ QI AfiQNDVKL APPTDVRSGY IRLVKNVNYY
DSfiS WVDW QfiL’Q VHE'DA VVNLDRGLYV YPEP<RYARS VQQYKILNCA
NYHLTQHQTD li'YDL'lr'WGQGL RAAP<KQKKH TLSLTPDTTT. YWAAQ CAN
YGKAFSVDK 4
DNA Sequence for PE from H. influenzae strain 772 (as set forth in: Microbes & ion,
Corrigendum to ”Identification of a novel Haemophi/us influenzae protein important for
adhesion to epithe/ia cells” [Microbes Infect. 10 (2008) 87-97], available online July 6, 2010,
”Article in ) - SEQ ID NO. 153
atgaaaaaaattattttaacattatcacttgggttacttactgcctgttctgctcaaatccaaaaggctaaacaaaatgatgtgaagctg
gcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggata
accaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgtt
ctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgc
gggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcga
gtgaagcattttcagttgataaaaaa
Protein Sequence for PE from H. influenzae strain 772 (as set forth in: Microbes & ion,
Corrigendum to ification of a novel Haemophilus influenzae protein important for
adhesion to epithe/ia cells” [Microbes Infect. 10 (2008) 87-97], available online July 6, 2010,
”Article in Press’)) - SEQ ID NO. 154
M<< T.TT.ST. GT.T.TACSAQI QKAKQNDVKL APPTDV’RSGY IRLVKNVNYY
DSfiS WVDW QfiL’Q VHE'DA VVNLDKGLYV YPEPKRYARS VQQYKILNCA
NY-ILTQVQTD GQGL RAAP<KQKKH TLSLTPDTTT. YWAAQ CAN
VDK <
Vector construction:
To generate LVL312, LVL291, LVL268, LVL269, LVL270, LVL702, , , LVL779,
LVL780, LVL781 and LVL782, a polymerase chain reaction (PCR) preparation of the following
components was prepared (specific components are subsequently exemplified): 36.6 pl of
deionized water, 5 pl of buffer #1 10X, 5 pl of dNTPs 2mM, 2 pl MgClz 25 mM, 0.4 pl of primer
#1 (50 pM), 0.4 pl of primer #2 (50 pM), 0.5 pl of template (100 ng/pl) and 0.4 pl of KOD HiFi
DNA polymerase 2.5 units/pl (NOVAGEN®) was formulated. Polymerase chain reaction involved
cycles of 15 seconds of denaturation at 98°C, 2 seconds for annealing at 55°C and 20
seconds of primer extension at 72°C. The PCR products were purified using QIAQUICK® PCR
purification kit (QIAGEN®). This product was used under conditions recommended by the
supplier which were: the addition of 5 volumes Buffer PB, provided in the QIAQUICK® PCR
purification kit, to 1 volume of the PCR preparation. The PCR preparation with Buffer PB was
subsequently mixed by vortex. A CK® column was placed into a 2 ml collection tube. To
bind DNA in the PCR preparation to the column, the mixed sample was applied to the
QIAQUICK ® column and centrifuged for 30—60 seconds at 14 000 RPM. The hrough was
discarded and the QIAQUICK ® column was placed back in the same tube. To wash the bound
DNA 0.75 ml Buffer PE, provided in the QIAQUICK ® PCR cation kit, was added to the
CK ® column, and the column was centrifuged for 30—60 seconds at 14 000 RPM. The
flow-through was discarded and the QIAQUICK ® column was placed back in the same tube.
The QIAQUICK ® column was centrifuged once more in the 2 ml collection tube for 1 minute to
remove residual wash buffer. Each QIAQUICK ® column was placed in a clean 1.5 ml
microcentrifuge tube. To elute the DNA, 33 pl water was added to the center of the QIAQUICK ®
membrane and the column was centrifuged for 1 minute at 14 000 RPM. Restriction enzymes
and buffer related were obtained from New England BioLabs. For example, approximately 5 pl
of pET26b vector (100 ng/pl), 2 pl of NEBuffer 2 (New England Biolabs, 1X NEBuffer 2: 50 mM
NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 at 25°C), 1 pl of Nde/ (20 000
units/ml), 1 pl of Hind/ll (20 000 units/ml) and 11 pl of deionized water were mixed and
incubated for two hours at 37°C for DNA digestion. Thereafter, a second step of purification was
performed using the QIAQUICK ® PCR purification kit (QIAGEN®) with the procedure described
above.
Ligation was performed using Quick T4 DNA ligase and Quick Ligation on Buffer from
New England s. For example, around 10 ng of vector and 30 ng of insert in 10 pl of
deionized water were mixed with 10 pl of 2X Quick Ligation Reaction Buffer (New England
Biolabs, 132 mM Tris-HCl, 20 mM MgClz, 2mM dithiothreitol, 2 mM ATP, 15% polyethylene
glycol, pH 7.6 at 25°C) and 1 pl of Quick T4 DNA ligase (New England s). The enzymatic
reaction was incubated for 5 s at room temperature before ormation.
To generate , LVL317, LVL318, LVL736, , LVL738, LVL739 and LVL740, a PCR
preparation of the following components was prepared: 40 pl of deionized water, 5 pl of reaction
buffer 10X, 1 pl of dNTPs mix, 1 pl of primer #1 (10 pM), 1 pl of primer #2 (10 pM), 1 pl of
te (25 ng/pl) and 1 pl of Pqu/tra High-Fidelity DNA polymerase 2.5 units/pl (QuikChange
ll Site-Directed Mutagenesis Kit, t Technologies, Stratagene Division) was formulated.
Polymerase chain on involved one cycle of denaturation at 95°C for 30 sec, 18 cycles of
sec of denaturation at 95°C, 1 min for annealing at 55°C and 5 min 30 sec of primer
extension at 68°C. The PCR products were digested using 1 pl of Dpnl restriction enzyme at
37°C for one hour before transformation.
A detailed list of PCR primer sequences used for amplifications is illustrated in Table 4.
To generate pRlT16711, the PE gene fragment coding for amino acids 22 to 160 of SEQ ID NO.
4, which excludes the sequence coding for its corresponding secretion signal, was amplified by
PCR from genomic DNA of NTHi strain 772. The amplification primers were designed based on
the available strain Hi Rd sequence (at that time, the 772 sequence was not . The 5’
primer sequence contains one mutation compared to the NTHi 772 sequence nce as now
available), introducing one amino acid difference in PE coding sequence at position 24, glutamic
acid (E) instead of lysine (K). After PCR amplification, the insert was cloned in the pET-26(+)
expression vector (NOVAGEN®) using BamHI and Xhol restriction sites.
To generate pRlT16671, a DNA nt coding for a PilA gene fragment (amino acids 40 to
149 of SEQ ID NO. 58, SEQ ID NO. 127), which excludes its leader peptide as well as a portion
of the predicted hobic alpha helix, was amplified from genomic DNA of NTHi strain 86-
028NP and cloned into the pET15 expression vector. The vector pRlT16790 (containing amino
acids 40 to 149 from NTHi strain 86-028NP) was used as a template to generate the vector
671. The PilA gene fragment was amplified by PCR using the vector pRlT16790 and
primers MDES PlLA-3 and MDES . The Pi/A fragment was cloned into the pET-26
expression vector using Nde/ / Xhol ction sites. The DNA sequence encoding six histidine
(his) amino acids was incorporated into the 5’ primer to add six histidines (6xhis) at the N-
terminal end of the PilA sequence (MDES PlLA-3).
To generate LVL312 (Flgl signal peptide-E-PilA fragment-GG-PE fragment-GGHHHHHH), a
polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 / strain
86-028NP) using the pRlT16671 vector as a template and s CAN534 and CAN537. DNA
sequence corresponding to Fig] signal peptide (sp) and glutamic acid (E) amino acid was
incorporated into the 5’ primer (CAN534). To link the PilA ce to PE sequence, DNA
sequence corresponding to the two glycine (GG) amino acids linker and the N-terminal PE
amino acids were incorporated into the 3’ primer 7). Another polymerase chain reaction
was performed to amplify the PE gene (amino acids 18-160) using pRlT16711 vector as a
te and primers CAN536 and CAN538. DNA ce corresponding to the C-terminal
PilA amino acids and GG amino acids were incorporated into the 5’ primer to link pilA to PE
sequence (CAN536). DNA sequence corresponding to the GG amino acids linker and 6xhis
amino acids were incorporated into the 3’ primer (CAN538). Finally, to generate LVL312, a third
polymerase chain reaction was performed to amplify the PilA and PE genes in fusion with the
Fig] signal peptide at the N-terminus, a glutamic acid (E) amino acid between Fig] and pilA, a
GG linker between PilA and PE sequences and a GG linker between PE and the 6xhis amino
acids at the C-terminus. To e this amplification, the products of the two polymerase chain
reactions described above were used as a template with primers CAN534 and CAN538. DNA
sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll
restriction site was incorporated into the 3’ . The generated PCR product was then
inserted into the pET-26b(+) cloning vector (NOVAGEN®).
To generate LVL291 (pelB signal peptide-PE fragment-GG-PilA fragment-GG-6xhis), a
polymerase chain reaction was performed to amplify the PE gene (amino acids ) using
the pRlT16711 vector as a template and primers CAN544 and CAN546. DNA sequence
corresponding to pelB signal peptide (sp) amino acids was incorporated into the 5’ primer
(CAN544). To link the PilA sequence to the PE sequence, DNA sequence ponding to GG
amino acids linker and the inal PilA amino acids were incorporated into the 3’ primer
(CAN546). Another polymerase chain reaction was med to amplify the PilA gene (amino
acids 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the pRlT16671 vector as a template
with primers CAN545 and CAN535. DNA sequence corresponding to the C-terminal PE amino
acids and GG amino acids were incorporated into the 5’ primer (CAN545) to link the PilA
sequence to the PE sequence. DNA ce corresponding to linker GG amino acids and
6xhis amino acids were incorporated into the 3’ primer (CAN535). Finally, to generate LVL291,
a third polymerase chain reaction was performed to amplify the PE and PilA genes in fusion with
the pelB signal peptide at the N-terminus, a GG linker between the PE and PilA sequences and
a GG linker between PilA and 6xhis amino acids at the C-terminus. To achieve this
amplification, the products of two polymerase chain reactions described above were used as a
template with primers CAN544 and CAN535. DNA sequence corresponding to Nde/ restriction
site was incorporated into the 5’ primer and Hind/ll restriction site was incorporated into the 3’
primer. The ted PCR product was then inserted into the pET-26b(+) cloning vector
(NOVAGEN®).
To generate LVL268 (pelB signal peptide-D-PE fragment-GG-PilA fragment-GG-6xhis), a
rase chain reaction was performed to amplify the PE gene (amino acids 20-160) using
the pRlT16711 vector as a template with primers CAN547 and CAN546. DNA sequence
corresponding to the pelB signal peptide (sp) amino acids and aspartic acid (D) amino acid were
incorporated into the 5’ primer (CAN547). To link the PilA ce to the PE sequence, DNA
sequence corresponding to GG amino acids linker and the N-terminal PilA amino acids were
incorporated into the 3’ primer (CAN546). Another polymerase chain reaction was performed to
y the PilA gene (amino acids 40-149 / NTHi strain 86-028NP) using the pRlT16671 vector
as a template with CAN545 and CAN535. DNA ce corresponding to the C-terminal PE
amino acids and GG amino acids were orated into the 5’ primer (CAN545) to link the PilA
sequence to the PE sequence. DNA sequence corresponding to linker GG amino acids and
6xhis amino acids were incorporated into the 3’ primer (CAN535). Finally, to generate LVL268,
a third polymerase chain reaction was med to amplify the PE and PilA genes in fusion with
the pelB signal peptide at the N-terminus, a D amino acid between pelB signal e and PE,
a GG linker between PE and pilA sequences and a GG linker between PilA and 6xhis amino
acids in C-term. To achieve this amplification, the products of the two polymerase chain
reactions described above were used as a template with primers CAN547 and CAN535. DNA
sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll
restriction site was incorporated into the 3’ primer. The generated PCR product was then
ed into the pET-26b(+) cloning vector (NOVAGEN®).
To generate LVL269 (NadA signal peptide-ATNDDD-PE nt-GG-PilA fragment-GG-6xhis),
a polymerase chain reaction was performed to amplify the PE gene (amino acids 22-160 of
SEQ ID NO. 4) using the pRlT16711 vector as a template with primers CAN548 and CAN546.
DNA sequence corresponding to pelB signal e (sp) amino acids and ATNDDD amino
acids were incorporated into the 5’ primer (CAN548). To link the PilA sequence to the PE
sequence, DNA ce corresponding to the GG amino acids linker and the inal PilA
amino acids were incorporated into the 3’ primer (CAN546). Another polymerase chain reaction
was performed to amplify the Pi/A gene (amino acids 40-149 of SEQ ID NO. 58, SEQ ID NO.
127) using the pRlT16671 vector as a te with primers CAN545 and CAN535. DNA
sequence corresponding to the C-terminal PE amino acids and GG amino acids were
orated into the 5’ primer to link the PilA sequence to the PE sequence (CAN545). DNA
sequence ponding to linker GG amino acids and 6xhis amino acids were incorporated into
the 3’ primer (CAN535). Finally, to generate LVL269, a third polymerase chain reaction was
performed to amplify the PE and PilA gene in fusion with the NadA signal peptide at the N-
terminus, ATNDDD amino acids between the pelB signal peptide and PE, a GG linker between
the PE and pilA ces and a GG linker between PilA and 6xhis amino acids at the C-
terminus. To e this amplification, the products of the two polymerase chain reactions
describe above were used as a template with primers CAN548 and CAN535. DNA sequence
corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction
site was incorporated into the 3’ primer. The generated PCR product was then inserted into the
pET-26b(+) cloning vector (NOVAGEN®).
To generate LVL270 (M-6xHis-PE fragment-GG-PilA fragment), a polymerase chain reaction
was performed to amplify the PE gene (amino acids 17-160) using the 711 vector as a
template with primers CAN540 and CAN542. DNA sequence corresponding to 6xhis amino
acids were incorporated into the 5’ primer (CAN540). To link the PilA sequence to the PE
sequence, DNA sequence corresponding to the GG amino acids linker and the inal PilA
amino acids were incorporated into the 3’ primer (CAN542). Another rase chain reaction
was performed to amplify the PilA gene (amino acids 40-149 / NTHi strain 86-028NP) using
pRlT16671 vector as a template with primers CAN541 and . DNA sequence
corresponding to the C-terminal PE amino acids and GG amino acids were incorporated into the
’ primer (CAN541) to link the PilA to the PE sequence. Finally, to generate LVL270, a third
polymerase chain reaction was performed to amplify the 6-his-PE-GG-PilA gene in fusion. To
achieve this amplification, the products of the two polymerase chain reactions describe above
were used as a template with s CAN540 and . DNA sequence corresponding to
Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction site was
incorporated into the 3’ primer. The generated PCR product was then inserted into the pET-
26b(+) cloning vector (NOVAGEN®).
To te LVL315 (pelB signal peptide-MD-PE fragment-GG-PilA fragment-GG-6xhis), a site-
directed mutagenesis was performed to change the N-terminal PE amino acid ce from
QIQ to MD using LVL291 as a template with primers CAN670 and CAN671 and the
QuikChange ll irected Mutagenesis Kit (Agilent Technologies, Stratagene Division).
To generate LVL317 (pelB signal peptide-PE fragment-GG-pilA fragment), a site-directed
mutagenesis was performed to incorporate a stop codon between the PilA gene and the DNA
sequence corresponding to GGHHHHHH amino acid residues (SEQ ID NO: 3) using LVL291 as
a template with primers CAN678 and CAN679 and the QuikChange ll Site-Directed
Mutagenesis Kit (Agilent logies, Stratagene Division).
To generate LVL318 (pelB signal peptide-MD-PE-GG-PilA), a site-directed mutagenesis was
performed to incorporate a stop codon between the PilA gene and the DNA sequence
corresponding to HH amino acid residues (SEQ ID NO: 3) using LVL315 as a
template with primers CAN678 and CAN679 and the QuikChange ll Site-Directed Mutagenesis
Kit (Agilent Technologies, gene Division).
To generate LVL702 (LVL291 AQ), a polymerase chain reaction was med using the
LVL291 vector as template and primers CAN1517 and CAN1518. Deletion of three nucleotides
corresponding to the amino acid Q at the position 23 on LVL291 ce was incorporated to
the 5’ primer. The only difference between LVL702 and LVL291 is the deletion of amino acid Q
at the position 23 on LVL291 sequence. NdeI and Hind/ll restriction sites were incorporated into
the 5’ and 3’ primers respectively. The generated PCR product was then inserted into the pET-
26b(+) cloning vector (NOVAGEN®).
To generate LVL735 (LVL317 AQ), a polymerase chain reaction was performed using the
LVL317 vector as template and primers 7 and CAN1519. Deletion of three nucleotides
corresponding to the amino acid Q at the position 23 on LVL317 sequence was incorporated to
the 5’ . The only difference between LVL735 and LVL317 is the deletion of amino acid Q
at the position 23 on LVL317 sequence. NdeI and Hind/ll restriction sites were incorporated into
the 5’ and 3’ primers tively. The generated PCR product was then inserted into the pET-
26b(+) cloning vector (NOVAGEN®).
To generate LVL736 (LVL291 + SA), a site-directed mutagenesis was performed to add amino
acids 8 and A between amino acid 22 and 23 on LVL291 sequence. LVL291 was used as
template with primers CAN1531 and 2 and the QuikChange ll Site-Directed
Mutagenesis Kit (Agilent Technologies, gene Division).
To generate LVL737 (LVL291 + A), a site-directed mutagenesis was performed to add amino
acid A n amino acid 22 and 23 on LVL291 sequence. LVL291 was used as template with
primers CAN1529 and CAN1530 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent
Technologies, gene Division).
To generate LVL738 (LVL291 AQIQ), a site-directed mutagenesis was performed to delete
amino acids Q, | and Q at positions 23 to 25 on LVL291 sequence. LVL291 was used as
template with primers CAN1523 and CAN1524 and the QuikChange ll Site-Directed
Mutagenesis Kit (Agilent logies, Stratagene Division).
To generate LVL739 1 AQIQK), a irected mutagenesis was performed to delete
amino acids Q, |, Q and K at positions 23 to 26 on LVL291 sequence. LVL291 was used as
template with primers CAN1525 and CAN1526 and the QuikChange ll irected
Mutagenesis Kit (Agilent logies, Stratagene Division).
To generate LVL740 (LVL291 AQIQKA), a site-directed nesis was performed to delete
amino acids Q, I, Q, K and A at positions 23 to 27 on LVL291 sequence. LVL291 was used as
template with primers CAN1527 and CAN1528 and the QuikChange ll Site-Directed
Mutagenesis Kit (Agilent Technologies, Stratagene Division).
To generate LVL778 (LVL736 A6xHis tag), LVL779 (LVL737 A6xHis tag), LVL780 (LVL738
A6xHis tag), LVL781 (LVL739 A6xHis tag) and LVL782 0 A6xHis tag) a polymerase
chain reaction was performed using the LVL736, LVL737, LVL738, LVL739 and LVL740 s
as template, respectively, with primers CAN1669 and CAN543. Deletion of 6xHis tag
corresponds to the amino acid sequence GGHHHHHH (SEQ ID NO. 3) at the C-terminal
sequences. This deletion was incorporated to the 3’ primer. NdeI and Hind/ll restriction sites
were incorporated into the 5’ and 3’ primers respectively. The generated PCR product was then
inserted into the b(+) cloning vector (NOVAGEN®).
Table 4: PCR primer sequences used for PE, PilA and PE-PilA ications
DNA Sequence
'—3'
CACACACATATGATTAAATTTCTCTCTGCATTAATTCTTCTACTGGTCACGACGG
CAN534 CGGCTCAGGCTGAGACTAAAAAAGCAGCGGTATCTG (SfiQ 3 NO. 155)
TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTGTGTGACACTTCCG
CAN535 CAAAAATTTGC (SfiQ 3 NO. 156)
TTTGCGGAAGTGTCACACAAGGCGGCGCGCAGATTCAGAAGGCTGAACAAAATGA
CAN536 TGT (SfiQ 3 NO. 157)
TTTTGTTCAGCCTTCTGAATCTGCGCGCCGCCTTGTGTGACACTTCCGC
CAN537 AAA (SfiQ 3 NO. 158)
TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTTTTTATCAACTGAA
CAN538 AATG (SfiQ 3 NO. 159)
CACACACATATGCACCACCACCACCACCACAGCGCGCAGAT TCAGAAGGCTGAAC
CAN540 ATGT (SiiQ :3 NO. 160)
CATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATC (s '.
CAN541 NO. 161)
GATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAATG (s:
CAN542 NO. 162)
CAN543 TGTGTGAAGCTTTTATTGTGTGACACTTCCGCAAA (S '. 3 NO.
CATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCG
CTGCCCAGCCGGCGATGGCCCAGATTCAGAAGGCTGAACAAAATGATGT (SI
CAN544 __D NO . l 64)
TCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATCTG (s:
CAN545 :3 NO. 165)
CAGATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAATGC (s:
CAN546 :3 NO. 166)
CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCG
CTGCCCAGCCGGCGATGGCCGATATTCAGAAGGCTGAACAAAATGATGT (SEQ
CAN547 __D NO . l 67)
CACACACATATGAAACACT T TCCATCCAAAGTACTGACCACAGCCATCCT TGCCA
CTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATAAGGCTGAACAAAA
CAN548 TGATG (SfiZQ :3 NO. 168)
CAN678 GGAAGTGTCACACAATAAGGCGGCCACCACCACC (S 4'.Q 3 NO. 2-71)
CAN679 GGTGGTGGTGGCCGCCTTATTGTGTGACACTTCC (S 4'.Q 3 NO. 2-72)
GATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCG
CAN1517 CTGCCCAGCCGGCGATGGCCATTCAGAAGGCTGAACAAAA(S*'.Q 3 NO. 205)
CAN1518 GGCCGCAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCC(S*'.Q 3 NO. 206)
CAN1519 GGCCGCAAGCTTTTATTGTGTGACACTTCC(S*'.Q 3 NO. 207 )
GCTGCCCAGCCGGCGATGGCCAAGGCTGAACAAAATGATGTG (8de :3 NO.
CAN1523 208)
CACATCATTTTGTTCAGCCTTGGCCATCGCCGGCTGGGCAGC (s:
CAN1524 209)
GCTGCCCAGCCGGCGATGGCCGCTGAACAAAATGATGTGAAGC
CAN1525 2 l 0)
WO 39225
GCTTCACATCATTTTGTTCAGCGGCCATCGCCGGCTGGGCAGC
CAN1526 2 :- 1)
GCTGCCCAGCCGGCGATGGCCGAACAAAATGATGTGAAGCTGG
CAN1527 2 3—2)
CCAGCTTCACATCATTTTGTTCGGCCATCGCCGGCTGGGCAGC
8 2 l 3 )
GCTGCCCAGCCGGCGATGGCCGCCCAGATTCAGAAGGCTGAAC
9 2 l 4)
GTTCAGCCTTCTGAATCTGGGCGGCCATCGCCGGCTGGGCAGC (S '.
CAN1530 215)
GCTGCCCAGCCGGCGATGGCCAGCGCCCAGATTCAGAAGGCTGAAC
CAN1531 NO. 216)
GTTCAGCCTTCTGAATCTGGGCGCTGGCCATCGCCGGCTGGGCAGC (S'.
CAN1532 NO- 217)
CAN1669 CACACACATATGAAATACCTGCTGCCGACC (S '. 3 NO. 218)
GAATTCCATATGCACCATCACCATCACCATACTAAAAAAGCAGCGGTATCTGAA
MDesPILA-
(S41Q 3 NO. 173)
MDesPILAGCGCCGCTCGAGTCATTGTGTGACACTTCCGC
(S '. 3 NO.
GCCCAGCCGGCGATGGCCCAGATCCAGAAGGCTGAACAAAATG
MnoNTHL (s:
44 175)
CATTTTGTTCAGCCTTCTGGATCTGGGCCATCGCCGGCTGGGC
MnoNTHL (s:
45 176)
Transformation
ichia coli BLR (DE3) or E. coli HMS (DE3) cells were ormed with plasmid DNA
according to standard methods with CaClz-treated cells. (Hanahan D. « Plasmid transformation
by Simanis. » ln Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.).
Briefly, BLR (DE3) or HMS174(DE3) competent cells were gently thawed on ice. Approximately
4pl of plasmid (10-100 ng) were mixed using 50-100 pl competent cells. Thereafter, this
formulation was incubated on ice for 30 min. To perform the transformation reaction, the
formulation was heat pulsed at 42°C for 45 seconds then incubated on ice for 2 minutes.
Approximately 0.5 ml of SOC medium (Super l broth with lite repression) was
added to the transformed cells and the cell culture was incubated at 37°C for one hour before
plating on Luria-Bertani (LB) agar with 50 ug/ml kanamycin. Around 100 pl of ormed cell
culture was plated and incubated overnight at 37°C.
BLR (DE3): BLR is a recA‘ derivative of BL21 (F— ompT hstB(rB— mB—) gal dcm (DE3). This
E. coli strain used for expression of recombinant proteins improves plasmid monomer yields and
may help stabilize target plasmids containing repetitive sequences or whose products may
cause the loss of the DE3 prophage. er, F.W. (1991) J. Mol. Biol. 219: 37—44). The
detailed genotype of E.coli BLR (DE3) has been published by N®. (F- ompT hstB
(rB- mB-) gal dcm recA)306::Tn10 (TetR) (DE3).
HMS174 (DE3): HMS174 s provide the recA mutation in a K—12 background. Like BLR,
these s may stabilize certain target genes whose products may cause the loss of the DE3
prophage. The detailed genotype of E.coli HMS174 (DE3) has been published by NOVAGEN®.
(F— recA1 hst(rK12— mK12+) (DE3) (Rif R ).
Production using BLR (DE3) and Characterization of His tagged constructs are described
in Example 3 through Example 6
Example 3: Protein expression using shake flask
Generally, one confluent agar plate inoculated with Escherichia coli BLR (DE3) transformed with
recombinant plasmid was stripped, resuspended in culture media and used to inoculate 800 ml
of LB broth (Becton, Dickinson and Company) 1r 1% (weight/volume, w/v) glucose (Laboratoire
MAT, catalogue number: GR-0101) and 50ug/ml kanamycin (Sigma) to obtain O.D.600nm
between 0.1 and 0.2. Cultures were incubated at 37 °C with agitation of 250 RPM to reach an
0nm Of ~0..8
One ml of each culture was then collected, centrifuged at 14 000 RPM for 5 minutes and
supernatants and pellets were frozen at -20°C separately.
At an O.D.600nm ~0.8, the BLR (DE3) cultures were cooled down (-20°C, 20 minutes or 4°C, 1
hour, preferably at 4°C for 1 hour) before inducing the expression of the recombinant protein by
addition of 1 mM isopropyl B-Dthiogalactopyranoside (IPTG; EMD Chemicals Inc, catalogue
number: 5815) and incubation overnight at 16, 22 and 30°C, or 3 hours at 37°C with agitation of
250 RPM, preferably overnight at 22°C. After the induction period the cultures were centrifuged
at 14 000 RPM for 5 minutes or 6 000 RPM for 15 minutes and supernatant (media on
sample) and pellets ining soluble and insoluble fractions) were frozen at -20°C separately.
These ions are used for periplasmic protein expression.
Example 4: Protein purification using shake flask, cell pastes, His tagged constructs
Each bacterial pellet obtained after induction was resuspended in 20 mM ydroxyethyl)
piperazineethanesulfonic acid ) buffer (pH 8.0) ning 500 mM NaCl, 10 mM
imidazole and Roche COMPLETE® Protease tor Cocktail (1 tablet/50 ml of HEPES buffer
containing 500 mM NaCl, Roche COMPLETE® ULTRA tablets, Roche Diagnostics
Corporation).
Alternatively, 20 to 50 mM bicine buffer may be used instead of HEPES buffer containing NaCl.
For example, 20 mM bicine buffer may be used. Bacteria were lysed using a Constant System
1.1 KW 2 X 30 000 PSI (pounds per square inch). Soluble natant) and insoluble (pellet)
components were separated by centrifugation at 20 000g for 20 min at 4°C.
6-His tagged-proteins were purified under native ions on immobilized metal affinity
chromatography (IMAC) using PROFINIATM protein purification protocol (Bio-Rad Laboratories,
Inc.) The soluble components were loaded on a 5m| His Trap column (Bio-Rad Laboratories,
Inc.) preequilibrated with the same buffer used for ial resuspension; the soluble
components were added at up to 5 ml/min (producing a “flow through fraction”) After loading on
the column, the column was washed with 10 column volumes of the same buffer at a rate of 10
ml/min (producing a “wash fraction #1). A second wash using 20 mM bicine buffer or 20 mM
HEPES buffer (pH 8.0) containing 500 mM NaCl and 20 mM imidazole was performed,
producing a “wash fraction #2). Elution was med using 2 column volumes of 20mM
HEPES buffer or 50mM bicine buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole
at a rate of 10 ml/min, ing an “elution fraction”.
To improve the purity of the n, positive elution fractions from IMAC were pooled and
loaded on a size exclusion chromatography (SEC) column DTM SUPERDEXTM 200 26/60
from GE Healthcare) preequilibrated in phosphate buffered saline without calcium or
magnesium (NaCl 137 mM, KCI 2.7 mM, NazHPO4 8.1 mM, KH2PO4 1.47 mM, pH 7.4).
s from elution ons were ed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Samples were trated using Centricon 10 000 MW
(Millipore).
Protein concentration was determined using spectrometer.
Example 5: SDS-PAGE and Western Blot Analzsis of His tagged constructs
& SDS-PAGE is of non-his tagged LVL317 & LVL318 constructs
e and insoluble fraction preparation
For example, 1 ml of culture after induction (see, for example, Example 3 above) was
centrifuged at 14 000 RPM for 2 min. The pellet was resolubilized using 40 pl of BUGBUSTER®
Protein Extraction Reagent (NOVAGEN®, EMD4 Biosciences, Merck), creating a cell
suspension. The cell suspension was incubated on a rotating platform for 10 min at room
temperature. The cell suspension was then centrifuged at 14 000 RPM for 2 min to separate the
soluble fraction. The resulting pellet (insoluble fraction) was resolubilized using 70 pl of
deionized water, 5 pl of dithiothreitol (DTT) 1M and 25 pl of NUPAGE® LDS (Lithium Dodecyl
Sulphate) Sample Buffer 4X (INVITROGENTM). The soluble fraction (supernatant from the cell
suspension of the resolubilized pellet) was added to 30 pl of deionized water, 5 pl of DTT 1M
and 25 pl of LDS Sample Buffer 4X.
Media on preparation
For example, to prepare the media fraction, 100 pl of the atant from the induced whole
cell culture following centrifugation (see, for example, Example 3 above) was concentrated by
adding 500 pl of RC reagent l (Bio-Rad Laboratories, Inc); the sample was mixed and
incubated for 1 min at room ature. Then, 500 pl of Reagent ll (Bio-Rad Laboratories, Inc.)
was added to the sample and mixed. This formulation was centrifuged at 14 000 RPM for 10
min. The pellet was resolubilized using 28 pl of deionized water, 2 pl of DTT 1M and 10 pl of
LDS SB 4X.
Purification fraction preparation
For example, purified proteins (for example, obtained as described in Example 4) were prepared
for SDS-PAGE analysis by adding 70 pl of sample, 5 pl of DTT 1M and 25 pl of LDS Sample
Buffer 4X.
SDS—PAGE analysis and transfer to nitrocellulose membrane
SDS—PAGE analysis and transfer to nitrocellulose membrane were performed according to
manufacturer’s endations (lnvitrogen) using NUPAGE® Bis-Tris 4-12% gels.
Preparations of samples, buffers and migration conditions were done under conditions
recommended by the ers.
In one example, the gel was loaded with a 20 ul sample from a master mix comprising 70 pl of a
ed protein fraction, 5 pl of DTT 1M and 25 pl of LDS SB 4X.
After samples were run on NUPAGE® Bis-Tris 4-12% gels, the proteins were transferred to
nitrocellulose membranes.
Nitrocellulose membranes were d for 30 minutes at 37°C, 60 RPM using 3 % milk/ PBS
1X fresh solution. After the ng incubation, Primary Antibodies were added (6X His Tag®
antibody, Abcam PLC, catalogue number: ab9108) at a dilution of: 1:1000 in 3 % milk/ PBS 1X
fresh solution for 1 hour at 37°C, 60 RPM. After that, membranes were washed three times, for
minutes each, at room temperature using 0.02% polsorbate 20 (for example, TWEENTM 20) /
PBS 1X. Secondary Antibodies ine phosphatase (AP) Rabbit anti-IgG (H+L) rabbit,
Jackson Research Laboratories, Inc.) were added at dilution 1:14 000 using 3 % milk/
PBS 1X fresh solution. Membranes were incubated for 1 hour at 37°C, 60 RPM. After that,
membranes were washed three times for 5 s at room ature using 0.02%
rbate 20 (for example, TWEENTM 20) / PBS 1X before the membrane expositions to 5-
bromochloro-3—indolyl phosphate/nitro blue tetrazolium (for example, BCIP®lNBT from Sigma-
Aldrich®, 1 tablet/ 10 ml water).
See Figure 1 for SDS-PAGE of induced bacterial extracts for fusion protein constructs LVL291,
LVL268 and LVL269. Insoluble fraction (I), Soluble fraction (S) and Culture Media fraction (M)
were loaded for LVL291, LVL268 and LVL269 before and after induction (ind).
See Figure 2 for SDS-PAGE and Western blot related to purification extracts for fusion n
constructs LVL291, LVL268 and LVL269. Flow through fraction (Ft), Wash fraction (W) and
Elution fraction (E) were loaded for purification of LVL291, LVL268 and . Anti-his tag
was used to probe extracts.
See Figure 3 for SDS-PAGE of induced ial and purification extracts for fusion protein
constructs LVL291 and LVL315. Culture Media fraction (M), Soluble on (Sol), Insoluble
on (Ins), Flow through fraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and
Elution on (E) were loaded for LVL291 and LVL315.
See Figure 4 for SDS-PAGE of induced bacterial and purification extracts for fusion protein
construct LVL312. Culture Media fraction (M), Soluble fraction (Sol), Insoluble fraction (Ins),
Flow Through fraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction
(E) were loaded for LVL312.
See Figure 25 for SDS-PAGE of soluble fractions from induced bacterial extracts for fusion
protein constructs LVL291, LVL702, LVL736, LVL737, LVL738, LVL739, LVL740 and pET26b
vector (negative control). (a) ment 1 (b) Experiment 2 (c) Experiment 3. PE-PilA fusion
protein indicated by arrow.
See Figure 26 for the e band percentage of fusion protein in the soluble fraction from
Experiments 1, 2 and 3.
LVL317 and LVL318 bacterial extracts used in the SDS—PAGE analysis in Figure 5 and Figure
6, respectively, were ed generally as described above.
Figure 5. SDS-PAGE of induced (1mM and 10uM IPTG) bacterial extracts for fusion protein
construct LVL317. ts from before (NI) and after induction (In), Soluble fraction (S),
Insoluble fraction (I).
Figure 6. SDS-PAGE of induced (1mM and 10uM IPTG) bacterial extracts for fusion protein
construct LVL318. Extracts from before (NI) and after induction (In), Culture Media fraction (M),
Soluble fraction (S), Insoluble fraction (I).
Proteins separate by SDS-PAGE were transferred to an lmmobilon-P membrane. The
Coomassie Blue stained protein bands were cut and placed in a sequenator reactor.
cing was carried out ing to manufacturer’s protocol using an Applied tems
PROCISE® Protein Sequencer, model 494-cLC.
Table 5: Shake flask protein expression profiles and signal peptide cleavage for fusion protein
constructs.
Fusion ption Protein Signal
Protein N-term —> C-term Expression peptide
Construct profile cleavage
F|g| sp — E — PiIA fragment — GG — PE fragment —
LVL312 med
GGHHHHHH
LVL291 Confirmed
PeIB sp — D — PE fragment — GG — PiIA fragment —
LVL268 Confirmed
GGHHHHHH
NadA sp — ATNDDD — PE fragment — GG — PiIA fragment —
LVL269 Confirmed
GGHHHHHH
LVL270 MHHHHHH — PE fragment — GG — PiIA fragment : Not tested
PeIB sp — MD — PE fragment — GG — PiIA fragment —
LVL315 Confirmed
GGHHHHHH
LVL317 PeIB — PE fragment — GG — PiIA fragment : Confirmed
LVL318 PeIB sp — MD — PE fragment — GG — PiIA fragment
LVL702 PeIB sp — PE fragment — GG — PiIA fragment — GGHHHHHH : Confirmed
LVL736 PeIB sp — PE fragment — GG — PiIA nt — GGHHHHHH : Confirmed
LVL737 PeIB sp — PE fragment — GG — PiIA fragment — GGHHHHHH : Confirmed
LVL738 PeIB sp — PE nt — GG — PiIA fragment — GGHHHHHH : Confirmed
LVL739 PeIB sp — PE nt — GG — PiIA fragment — GGHHHHHH : Confirmed
LVL740 PeIB sp — PE fragment — GG — PiIA fragment — GGHHHHHH : Confirmed
So = Soluble fraction. In = Insoluble fraction. Se = Protein Secreted in the media fraction. Nt =
Not tested. The ing rating were based on a visual inspection (coomassie) + : low
expression; ++ : medium expression; +++ : high expression; - : no sion
Exam Ie 6: LVL291 Fusion rotein characterization
PHYSICAL PROPERTIES OF LVL291: Foldin of PE and Pi/A in LVL291 & Melt/n Point
Circular Dichromism .'
Analysis of Secondary Structure
Circular dichroism (CD) is used to determine the secondary structure composition of a protein
by measuring the difference in the tion of left-handed polarized light versus right-handed
polarized light which is due to structural asymmetry. The shape and the magnitude of the CD
spectra in the far-UV region (190-250nm) are different whether a protein exhibits a beta-sheet,
alpha-helix or random coil structure. The relative abundance of each secondary structure type in
a given protein sample can be calculated by comparison to reference spectra.
Far UV spectra are measured using an l path of 0,01cm from 178 to 250nm, with a 1nm
resolution and bandwidth on a Jasco J-720 spectropolarimeter. Temperature of the cell is
maintained at 23°C by a Peltier thermostated RTE-111 cell block. A nitrogen flow of 10L/min is
ined during the measurements.
Results:
The far-UV CD spectra obtained for PE (from construct pRlT16762), PilA (from construct pRlT
16790) and PE-PilA proteins are characteristic of folded proteins containing a mix of alpha and
beta structures, but PE is significantly richer in alpha helix than PilA and PE-PilA (Figure 7, CD
spectra of PE, PilA and PE-PilA fusion proteins).
In order to evaluate the integrity of the folding of PE and PilA individual ns once bound
together in a chimeric protein and then verify a possible interaction between both, difference
a were calculated.
. When the PE and PilA far-UV spectra are combined, the resulting spectrum superposes
to the um of PE-PilA chimer (Figure 8, Combination of PE and PilA CD um).
This result suggests that the PE-PilA chimer contains all the secondary structures that
are detected in the individual components. It also suggests that the fusion of the ns
has no major impact on the ary ures of the dual components and
consequently that the folding of PE and PilA is not significantly different whether the
proteins are separate or in fusion.
Melting Point Evaluation:
In order to evaluate if the expression in fusion has an impact on the thermodynamic properties
of the individual proteins, the melting points of PE, PilA and PE-PilA have been evaluated by
monitoring the defolding of the alpha helix with temperatue by circular ism.
The presence of alpha helix is characterized by a m in the Circular dichroism signal at
222nm, so a significant increase in CD signal at 222nm during temperature increase is an
indication of protein denaturation. The determination of the temperature at which the protein
undergoes loss in secondary structure allows the determination of the melting point (Tm), which
corresponds to the temperature at which half of the proteins have lost their structure.
Melting point can be determined by identification of the inflexion point on the l
denaturation curve obtained from a temperature versus CD 222nm plot.
. Melting point of PilA and PE as determined by far-UV CD are respectively of 52°C and
68°C (Figure 9, PilA thermal denaturation curve; Figure 10, PE thermal ration
curve).
. The PE-PilA fusion protein exhibits two distinct Tm’s at 48°C and 71°C (Figure 11, PE-
PilA fusion protein thermal denaturation curve). Those values te that the PE and
PilA proteins are still independently folded when bound into a chimer and that they
defold at a similar temperature whether they are separate or in fusion. The ation
that the defolding of the PilA portion at 48°C doesn’t cause precipitation or impact the
Tm of the PE portion at 71°C is a strong indication that the interaction between PE and
PilA within the fusion is minimal and that they don’t have a major observable impact on
each other. The melting points of proteins are sensitive to various external conditions,
including buffer ition or presence of interacting molecules; that no major ion
is observed upon fusion of PE and PilA is a strong indication of the preservation of most
of the structure and of the properties of both PE and PilA when they are bound together.
Example 7: Fermentation process
Fusion proteins of the invention may be prepared by methods known by those skilled in the
art.
Example 8: Protein Purification of PEI PiIAI and LVL317
PE protein purification from pR/T16762:
To generate the 762 expression , the pRlT16711 vector was digested using
BamHI and Ncol restriction enzymes in order to delete 6 amino acid residues between the
signal sequence (pelB) and PE. The vector obtained was named pRlT16712. In this vector,
there are 3 amino acids between the signal sequence pelB and PE: MDP. In a second step, a
site directed nesis was performed to change amino acid sequence from MDP to QIQ
using pRlT16712 as template with primers MnoNTHi-44 and MnoNTHi-45 (described in Table
4) and the QuikChange ll irected nesis Kit (Agilent Technologies, Stratagene
Division).
Working seed of E. coli BLR(DE3) containing PE QIQ (from the pRlT16762 construct) was
thawed from -80°C and used to prepare 100 ml of pre-culture in LB broth by overnight
incubation at 37°C under agitation at 215 RPM. After overnight incubation, eight flasks
containing 800 ml of LB APS were inoculated with 12.5 ml of pre-culture and ODeoo measured
at around 0.06. The cultures were incubated 3h at 37°C with shaking. At a ODeoo of around
0.9, 1mM IPTG was added to start the induction. During the induction, the cultures were
incubated 19h at 22°C with shaking. After induction, ODeoo was at around 2.2. The cell
cultures were transferred into 1L centrifuge bags placed inside 1L s and centrifuged at
4°C for 30 minutes at 6,000xg and atant discarded. 1m| aliquots of culture pre- and
post-induction and supernatant were kept for future is.
Lysis of the BLR(DE3) induced with PE QIQ
The centrifuge bags were d from the centrifugation bottles, opened and the pellet was
expulsed from the bag into a beaker. The eight pellets were pulled together and resuspended
in 100ml of binding buffer (20mM Hepes, 10mM imidazole, 500mM NaCl, pH 8.01). The Eco/i
BLR (DE3) ning the PE QIQ contruct were disrupted with the TS Series Bench Top cell
disrupter from Constant s Ltd. (1x30 szi; 1x15szi). The lysate was centrifuged 30
minutes, 6000RPM, 4°C. The supernatant was kept and loaded on an IMAC column.
IMAC purification of PE QIQ
IMAC column (BioRad, Bio-Scale Mini Profinity IMAC cartridge 5ml) was brated with 5CV
of Binding buffer (20mM HEPES, 10mM imidazole, 500mM NaCl, pH 8.01) at 5ml/min. 100ml
of lysate supernatant was loaded on the IMAC at 2.5mL/min. Flow-through was collected in
50ml fractions for future analysis. The column was washed with 3CV of Binding buffer to
remove unbound protein. Sample containing unbound proteins was collected in one aliquot of
ml in a 50 ml tube. The column was washed with 2CV of Wash buffer (20mM HEPES,
20mM imidazole, 500mM NaCl, pH 8.01) collected in 2 ml fractions in a 96 well plate. The
bound protein was then eluted with 6CV of 100% Elution buffer (20mM HEPES, 250mM
imidazole, 500mM NaCl, pH 8.01). The eluted protein was collected in 2 ml fractions in 96-
well . Wash and elution were performed at n.
Size exclusion chromatography (SEC) on the IMAC pool of PE QIQ
SEC column (GE healthcare, HILOADTM 26/60 SUPERDEXTM 75 prep grade, 60cm height
approx 319ml volume) was equilibrated with 3CV of SEC buffer (20mM HEPES, 150mM NaCl,
pH8.49). 11 ml of IMAC eluate was loaded onto the column at a flow rate of 2.5 . 2m|
fractions were collected from 0.3CV to 0.9CV. Two runs were performed then fractions were
analyzed by SDS-PAGE. Fractions from the two runs ning Prot E protein were pooled
together (“SEC pool” 48ml approx total volume). 500mM of ne was added to the SEC
pool.
Dosage of the PE QIQ poo/ed samples generated in the above SEC protocol
The SEC pool was dosed with the RCDC (Reducing Agent and ent Compatible)
method from the Bio-Rad RC DCTM kit following manufacturer’s protocol:
For each tested sample and standard, 25uL was distributed in uge tubes in duplicate.
125uL of d RC Reagent l was added into each tube; each tube was vortexed and
incubate for 1 minute at room temperature. 125uL of Bio-Rad RC Reagent II is added into
each tube; each tube is ed and then centrifuged at 14,000xg for 5 minutes.
Supernatants are discarded by inverting the tubes on clean, adsorbent tissue paper allowing
the liquid to drain completely from the tubes. 25.4uL of Reagent A (already prepared by
mixing 20uL of Reagent S per 1ml of Reagent A) is added to each tube; each tube is vortexed
and incubated at room temperature for 5 minutes, or until itate is completely dissolved.
Vortex before proceeding to next step. Add 200uL of DC reagent B to each tube and vortex
immediately. Incubate at room temperature for 15 minutes. Transfer all samples to a l
plate and read the adsorbance at 750nm to determine the protein tration for each
unknown protein sample.
The ProtE concentration was 1.069 mg/ml
PilA His-tagged protein purification:
PilA was purified following the general procedure below:
E. coli cells containing a construct encoding PilA or a fragment thereof are suspended in
BUGBUSTER® and BENZONASE® nuclease (NOVAGEN®), for example 10 ml
BUGBUSTER® and 10 ul BENZONASE® nuclease. The cell lysate is mixed at room
temperature on a rotating platform, for example, for 15 s. The cell lysate is centrifuged
at 4°C, for example at 16,000g for 20 minutes. The supernatant containing the protein is
added to a Ni NTA column containing Ni NTA HIS -B|ND® resin and mixed at 4°C, for
example for 1 hour. The column may consist of2 ml of Ni NTA HIS -B|ND® resin
(NOVAGEN®) and 10 ml 1X Binding Buffer (from NOVAGEN®’s Ni-NTA Buffer Kit). The
column flow through is then collected. The resin is washed two times with 1X wash buffer, for
example, containing 300 mM NaCl, 50mM NaHzPO4, 25 mM imidazone, pH 8.0). The wash is
ted by gravity flow. The protein is eluted from the column with 1X elution buffer, for
example, 300 mM NaCl, 50mM NaHzPO4, 250 mM imidazone, pH 8.0. The protein may be
further purified by dialysis with the Binding Buffer and rerun over a Ni NTA column as
described above.
Thrombin cleavage of PilA.
PilA is then incubated with thrombin (diluted 1/50) at room temperature for 16h, to remove the
histidine tag.
Size exclusion chromatography (SEC) on Pi/A cleaved with thrombin.
SEC column (GE healthcare, HILOADTM 26/60 EXTM 75 prep grade, 60cm height
approx 319ml volume) was equilibrated with 5CV of SEC buffer (20mM HEPES, 150mM NaCl,
pH8.52). Approximately 10 ml of cleaved PilA was loaded onto the column at a flow rate of
2.5 ml/min. 2m| fractions collected from 0.3CV to 0.9CV. Two runs were med then
ons were analyzed by SDS—PAGE. Fractions from the two runs ning cleaved PilA
protein were pooled together (“SEC pool”, 52ml approx total ).
Dosage of PilA, SEC pool.
The SEC pool was dosed with the RCDC method as described above. The cleaved PilA
concentration was at 5.37 mg/ml.
Dialysis of the PilA SEC pool with PBS 1x pH 7.4 (dialysis factor = 1600) and dosage by
RCDC
The concentration post-dialysis determined by RCDC was at 3.0 mg/ml.
Purification of L VL31 7
Osmotic shock
Since LVL317 fusion protein is expressed and processed in bacterial asm, the protein
was extracted by osmotic shock.
Frozen (-20°C) harvested E. coli B2448 cell paste containing LVL317 from 4 L of fermentor
culture were pooled and resuspended in a hypertonic buffer consisting of 24 mM Tris-HCl,
16% (w/v) sucrose, 9.9% (w/v) glucose, 10 mM EDTA, pH 8.0 up to a final volume of 4L. The
suspension was mixed gently for 30 min at room ature using a e propeller
installed on RW 16 basic stirrer, at medium speed. The suspension was centrifuged at 15,900
x g for 30 minutes at room temperature. Supernatant (SN1) was kept for gel analysis.
The resulting pellet was resuspended in a hypotonic solution; 38 mM MgClz, and mixed for 30
min at room temperature. The mixture was centrifuged at 15,900 x g for 30 minutes at room
temperature and the n recovered in the supernatant (SN2).
A clarification of the SN2 was performed by tion through a 0.45/0.2 um polyethersulfone
Sartorius Sartopore 2 MidiCap filter, at 600ml/min of flow rate.
The SN2 was d 1:3 with 20 mM NaHzPO4-Na2HPO4, pH 7.0, the pH adjusted to 7.0 if
necessary and another clarification by filtration h a 0.45/0.2 um polyethersulfone
Sartorius Sartopore 2 MidiCap filter, at 600ml/min was performed.
SP SEPHAROSETM Fast Flow (SP FF) chromatography
The diluted/filtered SN2 was loaded and captured on a strong ic exchanger resin (SP
SEPHAROSETM FF - GE Healthcare) in a 14 cm ID (internal diameter) x 20 cm length column
(column volume 3100ml) equilibrated with 2CV of 20 mM NaHzPO4/ NazHPO4 buffer pH 7.0.
After washing the column with 5CV of 20 mM NaHzPO4 / NazHPO4 buffer pH 7.0, the antigen
ined within LVL317) was eluted by increasing the concentration of NaCl up to 100 mM
in the same washing buffer.
See Figure 12 for a typical SP SEPHAROSETM Fast Flow chromatogram.
Q SEPHAROSETM Fast Flow (Q FF) chromatography
The antigen t in the SP FF Eluate was diluted 1:4 with a 20 mM Tris pH 8.5, pH
adjusted to 8.5 if ary and passed through a strong anionic exchanger resin (Q
SEPHAROSETM FF - GE Healthcare) in a 14 cm ID x 11.8 cm length column (column volume
) equilibrated with 2CV of 20 mM Tris buffer pH 8.5. The antigen was recovered in the
flow-through fraction.
See Figure 13 for a typical Q SEPHAROSETM Fast Flow chromatogram.
Concentration, diaflitration, rbate 80 addition and sterile filtration
The Q FF flow-through containing the antigen was trated up to 0.7-0.8mg/ml based on
chromatogram UV and diafiltered with 5DV of 10 mM KHZPO4 / KZHPO4 buffer pH 6.5 using a
Pellicon-2TM 10 kDa cutoff membrane (Millipore).
Using a 5% stock solution, polysorbate 80 (for example, TWEENTM 80) was added to the
ultrafiltration retentate and agitated for 30 minutes with magnetic r at 130rpm at 4°C. The
final concentration of polysorbate 80 was 0.04%. Ultrafiltration retentate was sterilized by
filtration through a 0.45/0.2 um Cellulose Acetate membrane (Sartobran 300, Sartorius). The
purified bulk was stored at —20°C or -80°C. Absolute protein concentration was measured by
AAA (Amino Acid is) at 0.737mg/ml.
Example 9: Use of Polysorbate 80
A titration experiment indicated that the addition of polysorbate 80, specifically, TWEENTM 80
to a final concentration of 0.04% (w/v) to the purified bulk prior to sterile filtration reduced
filamentous particle formation and aggregation.
According to DSC analysis, TWEENTM 80 d the degree of structural change °C)
seen after freeze/thaw cycles after storage at -20°C and after storage 4 days at 4°C, -20°C
and -80°C and 37°C.
e 10: SDS-PAGE and Western Blot is of LVL317
SDS-PAGE and Western Blot analysis:
NUPAGE®, Bis-Tris 4-12% gel was loaded as described below with 10ug of sample in NUPAGE® LD
sample buffer ning 50mM DTT heated 5min at 95°C (20uL of sample was loaded for samples
having low concentration). Migration: 35 minutes at ts at room temperature (RT) in NUPAGE(
MES Running Buffer. Gel Stained 2 hours in Instant blue (Novexin cat.: ISBO‘IL) and destained
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overnight in water.
Lane contents:
1: MW standard (10uL) 2: Start (total fraction) (10ug) 3: SN1 non filtered (10ug)
4: SN2 not ed (10ug) 5: Not extracted (10ug) 6: Load SP FF (10ug)
7: Flow through SP FF (6.9ug) 8: Wash SP FF (20uL) 9: Elution SP FF (10ug)
: Strip SP FF (10ug) 11: Load Q FF ) 12: Elution Q FF (9.8ug)
13: Strip Q FF (4.8ug) 14: TFF retentate before0.04% TWEENTM 80 spiked (10ug)
: Purified bulk Not filtered 0.04% TWEENTM 80 spiked (10ug)
16: Purified bulk e Filtered 0.04% TWEENTM 80 spiked (10ug)
17: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (20ug + spiked E. Coli Cell lysate Rix (H—
18: E. Coli Cell lysate Rix (2ug)
19: E. Coli Cell lysate Rix (1 pg)
: E. Coli Cell lysate Rix (0.5ug)
See Figure 14 for a SDS-PAGE of ln-process samples from purification process of PE-PilA fusion
protein.
For Western Blot, proteins were transferred at 4°C overnight at 30Volts in NUPAGE® transfer
buffer + 20% Methanol, 0.1% SDS on nitrocellulose membrane. Membranes were blocked 1
hour with 50mM Tris, 150mM NaCl pH 7.4 + 5% non—fat dry milk, incubated 2 hours in rabbit
onal primary antibody diluted in blocking buffer (anti-Prot—E 1/50 000 and anti-Ecoli
(BLR) 1/1 000), washed 3x5minutes in 50mM Tris pH 7.4 + 0.05% Tween 20, incubated 1
hour in secondary antibody (goat anti-rabbit conjugated to alkaline phosphatase diluted
1/5000 in ng buffer), washed 3x5minutes in wash buffer and ped in BClP/NBT
ate (1 tablet per 10ml). All incubations performed in 25ml per membrane.
See Figure 15 for a Western Blot of ln-process samples of cation process from PE-PilA
fusion protein. Blot using rabbit polyclonal anti-PE.
Lane contents:
1: MW standard (10uL) 2: Start (total on) (10ug) 3: SN1 non filtered (10ug)
4: SN2 not filtered (10ug) 5: Not extracted (10ug) 6: Load SP FF (10ug)
7: Flow through SP FF (6.9ug) 8: Wash SP FF (20uL) 9: Elution SP FF (10ug)
: Strip SP FF (10ug) 11: Load Q FF (8.9ug) 12: Elution Q FF (9.8ug)
13: Strip Q FF (4.8ug) 14: TFF retentate before0.04% TWEENTM 80 spiked (10ug)
: Purified bulk Not filtered 0.04% TWEENTM 80 spiked (10ug)
16: Purified bulk Sterile ed 0.04% TWEENTM 80 spiked (10ug)
17: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (20ug + spiked E. Coli Cell lysate Rix (H—
18: E. Coli Cell lysate Rix (2ug)
19: E. Coli Cell lysate Rix (1 pg)
: E. Coli Cell lysate Rix (0.5ug)
See Figure 16 for a Western Blot of cess samples of purification process from A
fusion n. Blot using rabbit polyclonal anti-Eco/i (BLR).
Lane contents:
1: MW standard (10uL) 2: Start (total fraction) (10ug) 3: SN1 non ed (10ug)
4: SN2 not filtered (10ug) 5: Not extracted (10ug) 6: Load SP FF (10ug)
7: Flow through SP FF (6.9ug) 8: Wash SP FF (20uL) 9: Elution SP FF (10ug)
: Strip SP FF (10ug) 11: Load Q FF (8.9ug) 12: n Q FF )
13: Strip Q FF (4.8ug) 14: TFF retentate before0.04% TWEENTM 80 spiked (10ug)
: Purified bulk Not filtered 0.04% TWEENTM 80 spiked (10ug)
16: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (10ug)
17: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (20ug + spiked E. Coli Cell lysate Rix (H—
18: E. Coli Cell lysate Rix (2ug)
19: E. Coli Cell lysate Rix (1 pg)
: E. Coli Cell lysate Rix (0.5ug)
SDS-PAGE and Western Blot figures comments: The PE-PilA fusion protein migrates at 30kDa.
The extraction by c shock extracts the fusion protein expressed and processed in bacteria
periplasm and reduced contamination from bacteria. Small loss of fusion protein during hypertonic
treatment (lane 3). A small proportion is not extracted by hypotonic treatment and s associate
with cells (lane 5). Small loss in SP FF Flow through (lane 7) and in strip fraction of both columns
(lanes 10 and 13). Since the total volume of strip fraction is low the loss of fusion protein is not
significant. Degraded bands are visible in strip fractions but not in final product. No significant
contamination from E. coli host cell proteins in purified bulk (lane 16).
Analysis of LVL735 and LVL778 yielded similar profiles as LVL317.
e 11 : Melting Point Data for PE, FHA and LVL317
Thermal transition of PE-PilA fusion non His-tagged protein (LVL317) was compared with the
thermal transition of both PE his-tagged (as described in Example 8) and cleaved PilA (as
described in Example 8) proteins, purified as described above.
Before DSC, PE and PilA were dialyzed overnight in 10mM K2HPO4/KH2PO4 pH 6.5 + 0.04%
Tween 80 (1:250 sample:buffer volume ratio) to have them in the same buffer as the fusion
protein. After dialysis, proteins concentration was measured by BCA and adjusted to
ml (PE) and 500ug/ml (PilA).
Analysis done on VPTM-DSC from MicroCal, LLC (part of GE Healthcare). The final dialysis
buffer was used as reference and cted from the scans. DSC scan rate 90°C/hr. In
order to evaluate the capacity to measure the thermal transition in the Final Container (FC)
after formulation, the fusion protein was diluted to the FC concentration (60ug/ml). Final
container data not shown.
Results:
See Figure 17 for Thermal transition of PE-PilA fusion protein and PE and PilA proteins.
: PilA (1), n E (Prot E, PE) (2), PE-PilA PB not diluted 737ug/ml (3), and PE-PilA
PB diluted at FC concentration 60ug/ml (4).
1 — PilA Tm: 53°C
2 — Protein E Tm: 63
3 — PE-PilA PB (Purified Bulk) not diluted 737ug/ml Tm1 : 537°C and Tm2: 661°C
4 — A PB diluted at FC concentration 60ug/ml Tm1: 532°C and Tm2: 67.6°C
Two transitions were detected in the ed fusion protein (LVL317) (curves 3 and 4).
The Tm1 (537°C) of the PE-PilA fusion protein is r to PilA transition (53°C).
Significant shift of Tm2 in PE-PilA (66.1°C) as ed to PE transition (63°C). The fusion of
both domains seems to ize the PE fragment.
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The shift of Tm2 in the diluted fusion protein as compared to undiluted is a concentration
artifact arising from the steep decreasing slope typical of aggregation which is tration
dependant
n g analysis of LVL735 and LVL778 were similar to that of LVL317.
Example 12: PE-PiIA fusion protein construct LVL291 anti-PiIA immunogenicity
response in Balb/c mice.
The immune se directed t purified LVL291 PE-PilA fusion protein (the LVL291
fusion protein without the heterologous signal peptide) formulated in ASO3A was evaluated in
Balb/c mice. Animals (20 mice/group) were immunized by the intramuscular route at days 0,
14 and 28 with 10 ug of PE (from vector pRlT16762), PilA (from vector pRlT16790) or PE-
PilA, each formulated in ASO3A. The control group was vaccinated with ASO3A alone. Antibody
response directed t each antigen was determined in individual sera collected at day 42.
No antibody response was obtained with the negative control. As shown in Figure 18, the
antibody response directed against PilA was higher in mice immunized with the PE-PilA fusion
compared to antibody response in mice immunized with monovalent PilA. The antibody
responses directed against PE were similar in mice immunized with the fusion protein and
mice immunized with monovalent PE. GMT = geometric means titer. Data were captured and
analyzed with the SOFTMAX® Pro Software (Molecular Devices) running under WINDOWS®
(Microsoft); the four ters logistic log function was used to calculate the standard curve.
The four-parameter logistic-log function describes, with a high degree of accuracy, the curve
of the reference serum ying a pronounced sigmo'idal shape when plotted on an optical
density-versus-concentration (log) scale. Antibody concentrations were calculated at each
dilution of mice serum samples by interpolation of the rd curve. The dy in quality
control sera and in n serum samples is obtained by averaging the values from all
dilutions that fall within the working range (10-80 %) of the dilution curve of the reference.
Results are shown in Figure 18, which graphs the antibody responses against LVL291 PE-
PilA fusion protein and t monovalent PE and PilA in the Balb/c mouse model.
2012/050236
Example 13: Murine nasopharzngeal colonization model. Immunization with PE-PiIA.
Challenge with NTHi strain 86-028NP and NTHi strain 3224A.
Balb/c female mice (20/group) were immunized intranasally at days 0 and 14 with 6pg of a
purified PE-PiIA fusion protein (LVL291 for challenge with NP; LVL317 for challenge
with strain 3224A) formulated with LT (heat labile toxin of Escheria coil) and on day 28 with 6
pg of a purified PE-PiIA fusion protein in phosphate buffered saline (PBS). Control mice
(20/group) were vaccinated with LT alone. Mice were subsequently challenged intranasally
with 5 x ‘106 CFU (colony forming units) of homologous NTHi strain 86-028NP and
logous NTHi strain 3224A. Homology and heterology are ined by reference to
the NTHi strain with which the mice were immunized. Bacterial colonies were counted in
nasal cavities removed 1 and 2 days after the challenge. D1 = day 1. D2 = day 2.
PE-PiIA vaccination increased the clearance of NTHi strain NP and strain 3224A in the
nasopharynx at day 1 and day 2 post challenge.
For the experiment performed with NTHi strain NP: A 2-way fixed ANOVA was
performed using the log10 values of the counts as response, the fixed factors being the
group (4 levels) and the day (2 levels). The assumption of variance heterogeneity was
rejected and a model with heterogeneous variances was fitted to the data. No significant
ction was detected between the 2 factors. The group fusion A (6 pg per
mouse) significantly reduced CFU compared with the control group (LT); the geometric
mean ratio being equal to 0.06 with a 95% confidence interval of 0.01, 0.25.
For the experiment conducted with NTHi strain 3224A: A 3-way fixed ANOVA was
performed using the log10 values as response, the fixed factors being the group, the day,
and the experiment. The Shapiro-Wilk and Levene’s test did not reject the assumptions of
ity and of homogeneity of variances. No significant interaction between any of the 2
factors or between the 3 factors was detected and only main factors were kept in the
analysis. PE-PiIA / LT significantly reduced CFU compared with the control group; the
ric mean ratio being equal to 0.11 with a 95% confidence interval of 0.02, 0.61.
See Figure 19 for effect of PE-PiIA fusion protein vaccination on NTHi strain 86-028NP
bacterial clearance in mouse nasopharynx.
See Figure 20 for effect of PE-PilA fusion protein ation on NTHi strain 3224A bacterial
clearance in mouse nasopharynx.
Example 14: Murine nasophamngeal colonization model. zation with FHA.
Challenge with NTHi strain 3219C.
Female OF1 mice (20 mice/group) were immunized asally at days 0 and 14 with 3pg
PilA (from vector 16790) formulated with LT and at day 28 with 3 ug PilA in PBS. Control mice
were vaccinated with LT alone. Mice were subsequently challenged intranasally with 5 x ‘106
CFU of NTHi strain 3219C. Bacterial es were d in nasal cavities removed 3 and
4 days after the challenge. D3 = day 3. D4 = day 4.
See Figure 21 for effect of PilA vaccination on bacterial clearance in mouse nasopharynx.
Example 15: Murine nasopharyngeal colonization model. Immunization with PE.
Challenge with NTHi strain 3224A.
Balb/c female mice (20 mice/group) were immunized intranasally at days 0 and 14 with 3pg
PE (from vector pRlT16762) formulated with LT and at day 28 with 3 ug PE in PBS. Control
mice were vaccinated with LT alone. Mice were subsequently challenged intranasally with 5 x
106 CFU of NTHi strain 3224A. Bacterial colonies were counted in nasal cavities removed 3
and 4 days after the challenge. 10 mice were examined on day 3 (D3). 10 mice were
examined on day 4 (D4). PE vaccination increased significantly the clearance of NTHi in the
naso-pharynx at day 4 post challenge (Figure 22), using on the Dunn test for statistical
analysis.
See Figure 22 for effect of PE vaccination on ial clearance in the nasopharynx of mice.
Example 16: Vibronectin binding. tion of Vibronectin binding by LVL317 &
LVL735 PE-PiIA fusion n.
The ability of PE in the purified LVL317 PE-PilA fusion protein construct to bind to vitronectin
was evaluated. Microtiter plates (POLYSORPTM, Nunc, Thermo Fisher Scientific) were coated
with PE (from vector pRlT16762) or with purified LVL317 PE-PilA fusion protein (10 ug/ml).
Plates were washed four times with NaCl polysorbate 20, 0.05% (for example,
TWEENTM 20) and d for one to two hours with PBS-BSA 1%. After four washings,
vitronectin (Vitronectin from human plasma, SlGMA-ALDRICH®) was added (10 ug/ml), two
fold diluted (12 dilutions), and the plates were incubated for 1h at room temperature. The
plates were then washed 4 times with NaCl 150mM-polysorbate 20, 0.05% (for example
TWEENTM 20) After washings, the bound vitronectin was detected using peroxydase sheep
anti-human ectin (US Biological) followed by the on of ortho-phenylene
diamine/H202 substrate. The color developed is directly proportional to the amount of antibody
fixed to the vitronectin.
See Figure 23 for (a) LVL317 PE-PilA fusion protein bound to vitronectin. PilA = PilA from
NTHi strain 86-028NP (as bed for pRlT16790); PE = Protein E (as described for
pRlT16762) and (b) LVL317 and LVL735 PE-PilA fusion protein bound to vitronectin.
Example 17: Vibronectin binding. Inhibition of Vibronectin binding by antibodies
directed against the LVL291 PE-PiIA fusion protein.
Microtiter plates (POLYSORPTM, Nunc, Thermo Fisher ific) were coated with PE (from
vector pRlT16762) or with purified PE-PilA fusion protein (10 ug/ml). Plates were washed four
times with NaCl 150mM-polysorbate 20, 0.05% (for example, TWEENTM 20) and blocked for
two hours with A 1%. After gs, vitronectin (Vitronectin from human plasma,
ALDRICH®) was added at 50ug/ml and purified antibodies anti-PE-PilA ced and
purified in house) were two-fold serially diluted and incubated for 1h at room temperature. The
plates were then washed 4 times with NaCl 150mM-polysorbate 20, 0.05% (for example,
TWEENTM 20). After four washings, the bound vitronectin was detected using peroxydase
sheep anti-Vitronectin (US Biological) followed by the addition of ortho-phenylene
e/H202 ate. The color developed is directly proportional to the amount of antibody
fixed to the vitronectin.
tion of vitronectin binding to PE by polyclonal antibodies directed against PE-PilA was
observed.
See Figure 24 for inhibition of vitronectin binding by polyclonal antibodies against PE-PilA
fusion protein.
e 18: Antigenicity of LVL291 PE-PiIA fusion protein. ELISA.
Purified LVL291 PE-PilA fusion protein was validated in an antigenicity test with lent
proteins as control. The fusion protein was tested in a sandwich ELISA developed with
polyclonal antibodies (rabbit and guinea pig) generated against the PE gene fragment coding
for amino acids 22 to 160 of SEQ ID NO: 4 (as described for pRlT16711) or against PilA from
NTHi strain 86-028NP (from vector pRlT16790).
PilA or PE was added at 100 ng/ml and serially two fold diluted. After 30 minutes incubation
and after washing, the bound antigen was detected by a rabbit polyclonal serum obtained
after immunisation with PE or PilA. The bound antibodies were detected using a peroxydase
anti-rabbit lg on lmmunoResearch Laboratories, Inc.) followed by the addition of ortho-
phenylene—diamine/HzOz substrate. The color developed is directly proportional to the amount
of antigen t. Absorbance readings were measured using a spectrophotometer for
microtiter plates. The antigenicity of the samples was ined by ison to the curve
of the full length PE or full length PilA reference n and is expressed in ug/ml. The
reference represented 100% of antigenicity.
As observed in the Table 6: Antigenicity was observed with the purified LVL291 PE-PilA fusion
protein compared to the monovalent PE and PilA antigens.
Table 6 : Relative antigenicity obtained with purified LVL291 PE-PilA fusion protein in the
antigenicity test.
—PErelative antigenicity (%)
Protein E as Reference 100
PE-PilA 130-148
—PErelative antigenicity (%)
PilA as Reference 100
PE-PilA 120-152
Example 19: Immunogenicity of LVL735 PE-PiIA fusion protein.
Female Balb/c mice (n = 34) were immunized by the uscular route at days 0, 14 and 28
with 50 pl of vaccine formulation containing 1, 0.2 or 0.04 pg of A fusion protein LVL317
or LVL735 formulated within ASO‘IE or AIPO4 (aluminium phosphate). The antibody responses to
2012/050236
PE and PilA were determined in individual sera collected at day 42 and the lgG level against PE
and PilA was measured and expressed in ug /m|.
See Figure 27 for PE and PilA antibody response to LVL317 and LVL735. GMC= geometric
mean concentration = geometric means titer.
. GMT IC = confidence als.
Example 20: Protective efficacy of the LVL735 and LVL317 fusion proteins in a mouse
model of Non-tzgeable hilus influenzae nasophamngeal colonization.
Female Balb/c mice were intranasally immunized at days 0 and 14 with 10 ul of vaccine
formulation containing 5.8 ug of LVL735 or LVL317 admixed with 0.5 ug of E. coli labile toxin
(LT). A r dose of 5.8 ug of non-adjuvanted LVL735 or LVL317 was administered at day
28. Control mice were vaccinated with LT alone at days 0 and 14, and PBS at day 28. Animals
were intranasally challenged with 5 x ‘106 cfu of NTHi 3224A strain at day 42. Bacterial colonies
were counted in nasal cavities removed 1 and 2 days after the challenge (n = 10/time-point).
Nasal cavities are homogenized in medium and a bacterial quantification is performed. Results
are well expressed in CFU/ml.
See Figure 28 for the effect of LVL735 and LVL317 vaccination on bacterial nce in a
mouse model of non-typeable Haemophi/us zae nasopharyngeal colonization.
Claims (47)
- CLAIMS 1. A fusion protein of formula I: (X) m – (R1)n – A – (Y) o – B – (Z)p (formula I) wherein: X is a signal peptide or MHHHHHH (SEQ ID NO. 2); m is 0 or 1; R1 is an amino acid; n is 0, 1, 2, 3, 4, 5 or 6; A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, or PilA from Haemophilus influenzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h n h is 4, 5, 6, 7, 8, 9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment thereof, or Protein E from Haemophilus nzae or an immunogenic fragment thereof; Z is HH (SEQ ID NO. 3); and p is 0 or 1, wherein when A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an immunogenic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an genic fragment thereof, B is not PilA from Haemophilus influenzae or an immunogenic fragment thereof.
- 2. A fusion n according to claim 1 wherein X is selected from the group consisting of FlgI, NadA and pelB.
- 3. A fusion protein according to any of claims 1-2 n m is 0.
- 4. A fusion protein according to any of claims 1-3 wherein n is 0.
- 5. A fusion protein according to any of claims 1-4 wherein A is an immunogenic nt of Protein E, wherein Protein E is selected from any one of SEQ ID NO. 4 – SEQ ID NO. 57.
- 6. A fusion protein according to any of claims 1-5 n A is the genic fragment of Protein E from H. nzae as set forth in SEQ ID NO: 124.
- 7. A fusion protein according to any of claims 1-6 wherein Y is GG.
- 8. A fusion protein according to any one of claims 1-7 wherein B is an immunogenic fragment of PilA, wherein PilA is selected from any one of SEQ ID NO. 58 – SEQ ID NO. 121.
- 9. A fusion protein according to any of claims 1-8 wherein B is the immunogenic fragment of PilA from H. influenzae as set forth in SEQ ID NO. 127.
- 10. A fusion protein selected from the group consisting of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO.182, SEQ ID NO.184, SEQ ID NO.186, SEQ ID NO.188, SEQ ID NO. 190, SEQ ID NO.192, SEQ ID NO.194, SEQ ID NO.196, SEQ ID NO.198, SEQ ID NO.200, SEQ ID NO.202 and SEQ ID NO.204.
- 11. A fusion protein approximately 95% cal to any of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 or SEQ ID NO. 204.
- 12. A fusion protein of claim 10 or claim 11 wherein the signal peptide has been removed.
- 13. The fusion protein of SEQ ID NO. 148, wherein the signal peptide has been d, SEQ ID NO. 177 (QIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK EPKR YARSVRQYKI LNCANYHLTQ DEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQ).
- 14. The fusion protein of SEQ ID NO. 194, wherein the signal peptide has been removed, SEQ ID NO. 219 (IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV EFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ).
- 15. An immunogenic composition comprising isolated Protein E from H. influenzae and isolated PilA from H. influenzae.
- 16. An immunogenic composition of claim 15 wherein Protein E is a polypeptide of SEQ ID NO. 4, a polypeptide comprising a sequence having at least 75%, 77%, 80%, 85%, 90%, 95%, 97%, 99% or 100% ty, over the entire length, to SEQ ID NO. 4, or is a polypeptide sing an genic fragment of at least 7, 10, 15, 20, 25, 30 or 50 uous amino acids of SEQ ID NO. 4.
- 17. The immunogenic composition of claim 16, wherein the immunogenic fragment comprises a B and/or T cell epitope of SEQ ID NO: 4.
- 18. The genic composition of claims 15-17 wherein Protein E is capable of eliciting an immune response which recognizes SEQ ID NO. 4.
- 19. An immunogenic composition of claims 15-18 n PilA is a ptide of SEQ ID NO. 58, a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 97% or 100% identity, over the entire length, to SEQ ID NO. 58, or is a polypeptide comprising an immunogenic fragment of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58.
- 20. The immunogenic composition of claim 19, n the immunogenic fragment comprises a B and/or T cell epitope of SEQ ID NO: 58.
- 21. An immunogenic composition of any of claims 15-20 wherein PilA is capable of eliciting an immune response which recognizes SEQ ID NO. 58.
- 22. The immunogenic composition of claims 15-21, wherein the Protein E from H. influenzae and the PilA from H. influenzae are comprised as the fusion protein of claims 1-14.
- 23. An immunogenic ition comprising the fusion protein of SEQ ID NO. 177 (QIQKAEQN DVKLAPPTDV LVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFW APKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA ELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQ).
- 24. An genic composition comprising the fusion protein of SEQ ID NO. 219 (IQKAEQND VKLAPPTDVR VKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG CKGT DASLFPANFC .
- 25. A vaccine comprising the fusion protein of any of claims 1-14 or genic compositions of any of claims 15-24.
- 26. The use of a fusion protein according to any one of claims 1 to 14, an immunogenic composition according to any of claims 15-24 or the vaccine of claim 25, in the manufacture of a medicament for the treatment or prevention of otitis media in a subject in need thereof.
- 27. The use according to claim 26, wherein the immunogenic composition is a composition according to claim 23 or claim 24.
- 28. The use of a fusion protein according to any one of claims 1 to 14, an immunogenic composition according to any of claims 15-24 or the vaccine of claim 25, in the manufacture of a medicament for the treatment or tion of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in a subject in need thereof.
- 29. The use according to claim 28, wherein the genic composition is a composition according to claim 23 or claim 24.
- 30. The use of a fusion protein according to any one of claims 1 to 14, an genic composition according to any of claims 15-24 or the vaccine of claim 25 in the manufacture of a medicament for the treatment or prevention of pneumonia in a subject in need thereof.
- 31. The use according to claim 30, wherein the immunogenic composition is a composition ing to claim 23 or claim 24.
- 32. The use of a fusion protein according to any one of claims 1 to 14, an immunogenic composition according to any of claims 15-24 or the vaccine of claim 25 in the manufacture of a medicament for the treatment or tion of a H. influenzae infection or disease.
- 33. The use according to claim 32, wherein the immunogenic composition is a composition according to claim 20 or claim 21.
- 34. The use of claim 32 or claim 33, wherein the H. nzae infection or disease is an NTHi infection or disease.
- 35. The fusion protein of claims 1-14, or the immunogenic composition of claims 15-24, or the vaccine of claim 25, for use in the treatment or prevention of otitis media.
- 36. The fusion protein, the immunogenic ition, or the vaccine of claims 1-25, 35, for use in the treatment or prevention of acute bations of chronic obstructive pulmonary disease D).
- 37. The fusion protein, the immunogenic composition, or the vaccine of claims 1-25, 35-36, for use in the treatment or prevention of pneumonia.
- 38. The fusion protein, the immunogenic composition, or the vaccine of claims 1-25, 35-37, for use in the treatment or prevention of H. influenzae infection or disease.
- 39. The fusion protein, the immunogenic composition, or the vaccine of claim 38, for use in the treatment or prevention of NTHi ion or disease.
- 40. A process for producing periplasmic expression of a fusion protein of any one of claims 1 to 14, wherein the process comprises inducing expression of proteins containing a signal peptide.
- 41. The process of claim 40 wherein the signal peptide is from FlgI.
- 42. The process of claim 40 wherein the signal e is from pelB.
- 43. A process for making a vaccine comprising the process of any one of claims 40-42.
- 44. A fusion protein according to any one of claims 1 to 14, substantially as herein bed or exemplified.
- 45. An immunogenic composition according to any one of claims 15 to 24, ntially as herein described or exemplified.
- 46. A vaccine according to claim 25, substantially as herein described or exemplified.
- 47. A use according to any one of claims 26-34, substantially as herein described or ified. 51. A process according to claim 40, substantially as herein described or exemplified.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161474779P | 2011-04-13 | 2011-04-13 | |
US61/474,779 | 2011-04-13 | ||
US201161534012P | 2011-09-13 | 2011-09-13 | |
US61/534,012 | 2011-09-13 | ||
PCT/CA2012/050236 WO2012139225A1 (en) | 2011-04-13 | 2012-04-12 | Fusion proteins and combination vaccines comprising haemophilus influenzae protein e and pilin a |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ615328A NZ615328A (en) | 2015-10-30 |
NZ615328B2 true NZ615328B2 (en) | 2016-02-02 |
Family
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